Halohydrin dehalogenases and related polynucleotides

ABSTRACT

The present invention relates to novel halohydrin dehalogenase polypeptides and the polynucleotides that encode them. These polypeptides are useful in the production of 4-substituted-3-butyric acid derivatives and vicinal cyano, hydroxyl substituted carboxylic acid esters. The invention also provides related vectors, host cells and methods.

This application is a continuation-in-part (CIP) of U.S. Ser. No.11/067,323, filed Feb. 23, 2005, now pending, which is acontinuation-in-part (CIP) of U.S. Ser. No. 10/917,179, filed Aug. 11,2004, now pending, which claims the benefit of U.S. Ser. No. 60/546,033,filed Feb. 18, 2004, and U.S. Ser. No. 60/494,382, filed Aug. 11, 2003,all of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to novel halohydrin dehalogenasepolypeptides and the polynucleotides that encode them.

BACKGROUND OF THE INVENTION

Halohydrin dehalogenase (“HHDH”), also named halohydrinhydrogen-halide-lyase or halohydrin epoxidase, [EC4.5.1] catalyzes theinterconversion of 1,2-halohydrins and the corresponding 1,2-epoxides:

U.S. Pat. No. 4,284,723 describes the use of a halohydrin epoxidase forthe production of propylene oxide. U.S. Pat. Nos. 5,166,061 and5,210,031 describe the use of this enzyme activity for the conversion of1,3-dichloropropanol (DCP) and epichlorohydrin (ECH) respectively to4-chloro-3-hydroxybutyronitrile (CHBN). HHDH enzymes from Agrobacteriumradiobacter and Corynebacterium have been characterized on a broad rangeof halogenated substrates (Van Hylckama Vlieg et al., J. Bacteriol.(2001) 183:5058-5066; Nakamura et al., Appl. Environ. Microbiol. (1994)60:1297-1301; Nagasawa et al., Appl. Microbiol. Biotechnol. (1992)36:478-482).

HHDH also catalyzes the ring opening of epoxides with nucleophiles otherthan chloride or bromide. It has been demonstrated that azide (N₃ ⁻),nitrite (NO₂ ⁻) and cyanide (CN⁻) can replace chloride in the opening ofepoxides (see Nakamura et al., Biochem. Biophys Res. Comm. (1991)180:124-130; Nakamura et al., Tetrahedron (1994) 50: 11821-11826; LutjeSpelberg et al., Org. Lett. (2001) 3:41-43; Lutje Spelberg et al.,Tetrahedron Assym. (2002) 13:1083):

Nakamura et al. (Tetrahedron (1994) 50: 11821-11826) describe the use ofHHDH for the direct conversion of DCP to chloro-3-hydroxy-butyronitrile(CHBN) through epichlorohydrin (ECH) as the intermediate:

Some halohydrin dehalogenases have been characterized. For example, HHDHfrom A. radiobacter AD1 is a homotetramer of 28 kD subunits.Corynebacterium sp. N-1074 produces two HHDH enzymes, one of which iscomposed of 28 kD subunits (Ia), while the other is composed of relatedsubunits of 35 and/or 32 kD (Ib). HHDH from some sources is easilyinactivated under oxidizing conditions in a process that leads todissociation of the subunits, has a pH optimum from pH 8 to 9 and anoptimal temperature of 50° C. (Tang, Enz. Microbial Technol. (2002)30:251-258; Swanson, Curr. Opin. Biotechnol. (1999) 10:365-369). Theoptimal pH for HHDH catalyzed epoxide formation has been reported as 8.0to 9.0 and the optimal temperature in the range of from 45° C. to 55° C.(Van Hylckama Vlieg et al., J. Bacteriol. (2001) 183:5058-5066; Nakamuraet al., Appl. Environ. Microbiol. (1994) 60:1297-1301; Nagasawa et al.,Appl. Microbiol. Biotechnol. (1992) 36:478-482). The optimal pH for thereverse reaction, ring opening by chloride, has been reported for thetwo Corynebacterium sp. N-1074 enzymes and is 7.4 (Ia) or 5 (Ib). Sitedirected mutagenesis studies on the A. radiobacter AD1 HHDH indicatedthat oxidative inactivation is due to disruption of the quartenarystructure of the enzyme by oxidation of cysteine residues (Tang et al.,Enz. Microbial Technol. (2002) 30:251-258).

Purified HHDH enzymes from different sources exhibit specific activitieson DCP ranging from 146 U/mg (Ib) to 2.75 U/mg (Ia) (Nakamura et al.,Appl. Environ. Microbiol. 1994 60:1297-1301; Nagasawa et al., Appl.Microbiol. Biotechnol. (1992) 36:478-482). The high activity of the lbenzyme is accompanied by a high enantioselectivity to produce R-ECH fromDCP, while the Ia enzyme produces racemic ECH.

HHDH encoding genes have been identified in Agrobacterium radiobacterAD1 (hheC), Agrobacterium tumefaciens (halB), Corynebacterium sp (hheAencoding Ia and hheB encoding lb), Arthrobacter sp. (hheA_(AD2)), andMycobacterium sp. GP1 (hheB_(GP1)). All enzymes have been functionallyexpressed in E. coli.

It is highly desirable for commercial applications of HHDH that theenzyme exhibits high volumetric productivity, that reactions run tocompletion in a relatively short period of time, with a high finalproduct concentration, with high enanantioselectivity, and that nochemical side products are formed. These characteristics of a processcan generally be used to define the broad characteristics of the enzyme:low Km for the substrate(s), high process stability, high specificactivity, no substrate and product inhibition under conditions wherechemical reactions are not proceeding. Currently available HHDH enzymesdo not fulfill all of these criteria. For instance, the conversion on1,2-epoxybutane and cyanide to 3-hydroxyvaleronitrile by HHDH proceedsat a maximum rate of 3 mmol/hr and this rate is sustained for only 10minutes (Nakamura et al., Biochem. Biophys Res. Comm. (1991)180:124-130). Conversion of DCP and ECH to4-chloro-3-hydroxybutyronitrile (CHBN) is also limited to rates of 2-3mmol/hr (Nakamura, U.S. Pat. Nos. 5,166,061 and 5,210,031). An in depthanalysis of the ECH to CHBN conversion reveals that while the hheBencoded HHDH-Ib enzyme has high activity, high productivity ismaintained for only 20 min after which further conversion occurs at arate that is at least 50-fold slower, with the overall conversion atjust over 60% (Nakamura et al. Tetrahedron (1994) 50: 11821-11826). Thedirect conversion of DCP, via ECH to CHBN proceeds at a reduced rate andresults in a 65.3% yield. Thus, HHDH as described in the literature doesnot meet the desired criteria for a catalyst in commercial applications.

Accordingly, new halohydrin dehalogenases would be highly desirable.

BRIEF SUMMARY OF THE INVENTION

The present invention has multiple aspects. In one embodiment, thepresent invention provides an isolated or recombinant HHDH polypeptidecomprising an amino acid sequence that is at least 80% identical to areference sequence selected from the group consisting of SEQ ID NOs: 2,4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76,78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136,138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164,166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192,194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220,222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248,250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276,278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304,306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332,334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360,362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388,390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416,418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444,446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472,474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500,502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528,530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556,558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584,586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612,614, 616, 618, 619, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638,640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666,668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694,696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722,724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750,752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772, 774, 776, 778,780, 782, 784, 786, 788, 790, 792, 794, 796, 798, 800, 802, 804, 806,808, 810, 812, 814, 816, 818, 820, 822, 824, 826, 828, 830, 832, 834,836, 838, 840, 842, 844, 846, 848, 850, 852, 854, 856, 858, 860, 862,864, 866, 868, 870, 872, 874, 876, 878, 880, 882, 884, 886, 888, 890,892, 894, 896, 898, 900, 902, 904, 906, 908, 910, 912, 914, 916, 918,920, 922, 924, 926, 928, 930, 932, 934, 936, 938, 940, 942, 944, 946,948, 950, 952, 954, 956, 958, 960, 962, 964, 966, 968, 970, 972, 974,976, 978, 980, 982, 984, 986, 988, 990, 992, 994, 996, 998, 1000, 1002,1004, 1006, 1008, 1010, 1012, 1014, 1016, 1018, 1020, 1022, 1024, 1026,1028, 1030, 1032, 1034, 1036, 1038, 1040, 1042, 1044, 1046, 1048, 1050,1052, 1054, 1056, 1058, 1060, 1062, 1064, 1066, 1068, 1070, 1072, 1074,1076, 1078, 1080, 1082, 1084, 1086, 1088, 1090, 1092, 1094, 1096, 1098,1100, 1102, 1104, 1106, 1108, 1110, 1112, 1114, 1116, 1118, 1120, 1122,1124, 1126, 1128, 1130, 1132, 1134, 1136, 1138, 1140, 1142, 1144, 1146,1148, 1150, 1152, 1154, 1156, 1158, 1160, 1162, 1164, 1166, 1168, 1170,1172, 1174, 1176, 1178, 1180, 1182, 1184, 1186, 1188, 1190, 1192, 1194,1196, 1198, 1110, 1112, 1114, 1116, 1118, 1120, 1122, 1124, 1126, 1128,1130, 1132, 1134, 1136, 1138, 1140, 1142, 1144, 1146, 1148, 1150, 1152,1154, 1156, 1158, 1160, 1162, 1164, 1166, 1168, 1170, 1172, 1174, 1176,1178, 1180, 1182, 1184, 1186, 1188, 1190, 1192, 1194, 1196, 1198, 1200,1202, 1204, 1206, 1208, 1210, 1212, 1214, 1216, 1218, 1220, 1222, 1224,1226, 1228, 1230, 1232, 1234, 1236, 1238, 1240, 1242, 1244, 1246, 1248,1250, 1252, 1254, 1256, 1258, 1260, 1262, 1264, 1266, 1268, 1270, 1272,1274, 1276, 1278, 1280, 1282, 1284, 1286, 1288, 1290, 1292, 1294, 1296,1298, 1300, 1302, 1304, 1306, 1308, 1310, 1312, 1314, 1316, 1318, 1320,1322, 1324, 1326, 1328, 1330, 1332, 1334, 1336, 1338, 1340, 1342, 1344,1346, 1348, 1350, 1352, 1354, 1356. 1358. 1360, 1362, 1364, 1366, 1368,1370, 1372, 1374, 1376, 1378, 1380, 1382, 1384, 1386, 1388, 1390, 1392,1394, 1396, 1398, 1400, 1402, 1404, 1406, 1408, 1410, 1412, 1414, 1416,1418, 1420, 1422, 1424, 1426, 1428, 1430, 1432, 1434, 1436, 1438, 1440,1442, 1444, 1446, 1448, 1450, 1452, 1454, 1456, 1458, 1460, 1462, 1464,1468, 1470, 1472, 1474, 1476, 1478, 1480, 1482, 1484, 1486, 1488, 1490,1492, 1494, 1496, 1498, 1500, 1502, 1504, 1506, 1508, 1510, 1512, 1514,1516, 1518, 1520, 1522, 1524, 1526, 1528, 1530, 1532, 1534, 1536, 1538,1540, 1542, 1544, 1546, 1548, 1550, 1552, 1554, 1556, 1558, 1560, 1562,1564, 1566, 1568, 1570, 1572, 1574, 1576, 1578, 1580, 1582, 1584, 1586,1588, 1590, 1592, 1594, 1596, 1598, 1600, 1602, 1604, 1606, 1608, 1610,1612, 1614, 1616, 1618, 1620, 1622, 1624, 1626, 1628, 1630, 1632, 1634,1636, 1638, 1640, 1642, 1644, 1646, 1648, 1650, 1652, 1654, 1656, 1658,1660, 1662, 1664, 1666, 1668, 1670, 1672, 1674, 1676, 1678, 1680, 1682,1684, 1684, 1686, 1688, 1690, 1692, 1694, 1696, 1698, 1700, 1702, 1704,1706, 1708, 1710, 1712, 1714, 1716, 1718, 1720, 1722, 1724, 1726, 1728,1730, 1732, 1734, 1736, 1738, 1740, 1742, 1744, 1746, 1748, 1750, 1752,1754, 1756, 1758, 1760, 1762, 1764, 1766, 1768, 1770, 1772, 1774, 1776,1778, 1780, 1782, 1784, 1786, 1788, 1790, 1792, 1794, 1796, 1798, 1800,1802, 1804, 1806, 1808, 1810, 1812, 1814, 1816, 1818, 1820, 1822, 1824,1826, 1828, 1830, 1832, 1834, 1836, 1838, 1840, 1842, 1844, 1846, 1848,1850, 1852, 1854, 1856, 1858, 1860, 1862, 1864, 1866, 1868, 1870, 1872,1874, 1876, 1878, 1880, 1882, 1884, 1886, 1888, 1890, 1892, 1894, 1896,1898, 1900, 1902, 1904, 1906, 1908, 1910, 1912, 1914, 1916, 1918, 1920,1922, 1924, 1926, 1928, 1930, 1932, 1934, 1936, 1938, 1940, 1942, 1944,1946, 1948, 1950, 1952, 1954, 1956, 1958, 1960, 1962, 1964, 1966, 1968,1970, 1972, 1974, 1976, 1978, 1980, 1982, 1984, 1986, 1988, 1990, 1992,1994, 1996, 1998, 2000, 2002, 2004, 2006, 2008, 2010, 2012, 2014, 2016,2018, 2020, 2022, 2024, 2026, 2028, 2030, 2032, 2034, 2036, 2038, 2040,2042, 2044, 2046, 2048, 2050, 2052, 2054, 2056, 2058, 2060, 2062, 2064,2066, 2068, 2070, 2072, 2074, 2076, 2078, 2080, 2082, 2084, 2086, 2088,2090, 2092, 2094, 2096, 2098, 2100, 2102, 2104, 2106, 2108, 2110, 2112,2114, 2116, 2118, 2120, 2122, 2124, 2126, 2128, 2130, 2132, 2134, 2136,2138, 2140, 2142, 2144, 2146, 2148, 2150, 2152, 2154, 2156, 2158, 2160,2162, 2164, 2166, 2168, 2170, 2172, 2174, 2176, 2178, 2180, 2182, 2184,2186, 2188, 2190, 2192, 2194, 2196, 2198, 2200, 2202, 2204, 2206, 2208,2210, 2212, 2214, 2216, 2218, 2220, 2222, 2224, 2226, 2228, 2230, 2232,2234, 2236, 2238, 2240, 2242, 2244, 2246, 2248, 2250, 2252, 2254, 2256,2258, 2260, 2262, 2264, 2266, 2268, 2270, 2272, 2274, 2276, 2278, 2280,2282, 2284, 2286, 2288, 2290, 2292, 2294, 2296, 2298, 2300, 2302, 2304,2306, 2308, 2310, 2312, 2314, 2316, 2318, 2320, 2322, 2324, 2326, 2328,2330, 2332, 2334, 2336, 2338, 2340, 2342, 2344, 2346, 2348, 2350, 2352,2354, 2356, 2358, 2360, 2362, 2364, 2366, 2368, 2370, 2372, 2374, 2376,2378, 2380, 2382, 2384, 2386, 2388, 2390, 2392, 2394, 2396, 2398, 2400,2402, 2404, 2406, 2408, 2410, 2412, 2414, 2416, 2418, 2420, 2422, 2424,2426, 2428, 2430, 2432, 2434, 2436, 2438, 2440, 2442, 2444, 2446, 2448,2450, 2452, 2454, 2456, 2458, 2460, 2462, 2464, 2466, 2468, 2470, 2472,2474, 2476, 2478, 2480, 2482, 2484, 2486, 2488, 2490, 2492, 2494, 2496,2498, 2500, 2502, 2504, 2506, 2508, 2510, 2512, 2514, 2516, 2518, 2520,2522, 2524, 2526, 2528, 2530, 2532, 2534, 2526, 2528, 2530, 2532, 2534,2536, 2538, 2540, 2542, 2544, 2546, 2548, 2550, 2552, 2554, 2556, 2558,2560, 2562, 2564, 2566, 2568, 2570, 2572, 2574, 2576, 2578, 2580, 2582,2584, 2586, 2588, 2590, 2592, 2594, 2596, 2598, 2600, 2602, 2604, 2606,2608, 2610, 2612, 2614, 2616, 2618, 2620, 2622, 2624, 2626, 2628, 2630,2632, 2634, 2636, 2638, 2640, 2642, 2644, 2646, 2648, 2650, 2652, 2654,2656, 2658, 2660, 2662, 2664, 2668, 2670, 2672, 2674, 2676, 2678, 2680,2682, 2684, 2686, 2688, 2690, 2692, 2694, 2696, 2698, 2700, 2702, 2704,2706, 2708, 2710, 2712, 2714, 2716, 2718, 2720, 2722, 2724, 2726, 2728,2730, 2732, 2734, 2736, 2738, 2740, 2742, 2744, 2746, 2748, 2750, 2752,2754, 2756, 2758, 2760, 2762, 2764, 2766, 2768, 2770, 2772, 2774, 2776,2778, 2780, 2782, 2784, 2786, 2788, 2790, 2792, 2794, 2796, 2798, 2800,2802, 2804, 2806, 2808, 2810, 2812, 2814, 2816, 2818, 2820, 2822, 2824,2826, 2828, 2830, 2832, 2834, 2836, 2838, 2840, 2842, 2844, 2846, and2848, wherein the amino acid sequence further comprises one or moreamino acid residues selected from the group consisting of V at position5, A at position 27, V at position 45, Q at position 46, I at position54, V at position 60, V at position 65, N at position 67, H at position72, I at position 77, S at position 87, R at position 91, T at position93, A at position 95, E at position 96, M or Q at position 100, T atposition 103, R or T at position 117, S at position 118, R or I or L orY at position 121, S at position 135, R at position 139+E at position176 or D at position 139+R at position 176, A at position 146, N atposition 153, H at position 166, G or A at position 177, M or C atposition 178, N or D at position 179, D or T at position 180, K atposition 181, N at position 182, V at position 190, A at position 199, Wor Y at position 201, Y at position 205, L at position 236, T or R atposition 238, F at position 239, V at position 246, E or A or S atposition 251, and P at position 252, wherein amino acid residue positionis determined by optimal alignment of the amino acid sequence of theHHDH polypeptide with the reference sequence.

HHDH polypeptides of the present invention include those encoded by anucleic acid that hybridizes under stringent conditions oversubstantially the entire length of a nucleic acid corresponding to areference nucleic acid sequence selected from the group consisting of:SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67,69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101,103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129,131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157,159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185,187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213,215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241,243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269,271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297,299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325,327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353,355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381,383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409,411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437,439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465,467, 469, 4712, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493,495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521,523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549,551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577,579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605,607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633,635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661,663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689,691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717,719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745,747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771, 773,775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801,803, 805, 807, 809, 811, 813, 815, 817, 819, 821, 823, 825, 827, 829,831, 833, 835, 837, 839, 841, 843, 845, 847, 849, 851, 853, 855, 857,859, 861, 863, 865, 867, 869, 871, 873, 875, 877, 879, 881, 883, 885,887, 889, 891, 893, 895, 897, 899, 901, 903, 905, 907, 909, 911, 913,915, 917, 919, 921, 923, 925, 927, 929, 931, 933, 935, 937, 939, 941,943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969,971, 973, 975, 977, 979, 981, 983, 985, 987, 989, 991, 993, 995, 997,999, 1001, 1103, 1005, 1007, 1009, 1011, 1013, 1015, 1017, 1019, 1021,1023, 1025, 1027, 1029, 1031, 1033, 1035, 1037, 1039, 1041, 1043, 1045,1047, 1049, 1051, 1053, 1055, 1057, 1059, 1061, 1063, 1065, 1067, 1069,1071, 1073, 1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091, 1093,1095, 1097, 1099, 1101, 1103, 1105, 1107, 1109, 1111, 1113, 1115, 1117,1119, 1121, 1123, 1125, 1127, 1129, 1131, 1133, 1135, 1137, 1139, 1141,1143, 1145, 1147, 1149, 1151, 1153, 1155, 1157, 1159, 1161, 1163, 1165,1167, 1169, 1171, 1173, 1175, 1177, 1179, 1181, 1183, 1185, 1187, 1189,1191, 1193, 1195, 1197, 1199, 1201, 1203, 1205, 1207, 1209, 1211, 1213,1215, 1217, 1219, 1221, 1223, 1225, 1227, 1229, 1231, 1233, 1235, 1237,1239, 1241, 1243, 1245, 1247, 1249, 1251, 1253, 1255, 1257, 1259, 1261,1263, 1265, 1267, 1269, 1271, 1273, 1275, 1277, 1279, 1281, 1283, 1285,1287, 1289, 1291, 1293, 1295, 1297, 1299, 1301, 1303, 1305, 1307, 1309,1311, 1313, 1315, 1317, 1319, 1321, 1323, 1325, 1327, 1329, 1331, 1333,1335, 1337, 1339, 1341, 1343, 1345, 1347, 1349, 1351, 1353, 1355, 1357,1359, 1361, 1363, 1365, 1367, 1369, 1371, 1373, 1375, 1377, 1379, 1381,1383, 1385, 1387, 1389, 1391, 1393, 1395, 1397, 1399, 1401, 1403, 1405,1407, 1409, 1411, 1413, 1415, 1417, 1419, 1421, 1423, 1425, 1427, 1429,1431, 1433, 1435, 1437, 1439, 1441, 1443, 1445, 1447, 1449, 1451, 1453,1455, 1457, 1459, 1461, 1463, 1465, 1467, 1469, 1471, 1473, 1475, 1477,1479, 1481, 1483, 1485, 1487, 1489, 1491, 1493, 1495, 1497, 1499, 1501,1503, 1505, 1507, 1509, 1511, 1513, 1515, 1517, 1519, 1521, 1523, 1525,1527, 1529, 1531, 1533, 1535, 1537, 1539, 1541, 1543, 1545, 1547, 1549,1551, 1553, 1555, 1557, 1559, 1561, 1563, 1565, 1567, 1569, 1571, 1573,1575, 1577, 1579, 1581, 1583, 1585, 1587, 1589, 1591, 1593, 1595, 1597,1599, 1601, 1603, 1605, 1607, 1609, 1611, 1613, 1615, 1617, 1619, 1621,1623, 1625, 1627, 1629, 1631, 1633, 1635, 1637, 1639, 1641, 1643, 1645,1647, 1649, 1651, 1653, 1655, 1657, 1659, 1661, 1663, 1665, 1667, 1669,1671, 1673, 1675, 1677, 1679, 1681, 1683, 1685, 1687, 1689, 1691, 1693,1695, 1697, 1699, 1701, 1703, 1705, 1707, 1709, 1711, 1713, 1715, 1717,1719, 1721, 1723, 1725, 1727, 1729, 1731, 1733, 1735, 1737, 1739, 1741,1743, 1745, 1747, 1749, 1751, 1753, 1755, 1757, 1759, 1761, 1763, 1765,1767, 1769, 1771, 1773, 1775, 1777, 1779, 1781, 1783, 1785, 1787, 1789,1791, 1793, 1795, 1797, 1799, 1801, 1803, 1805, 1807, 1809, 1811, 1813,1815, 1817, 1819, 1821, 1823, 1825, 1827, 1829, 1831, 1833, 1835, 1837,1839, 1841, 1843, 1845, 1847, 1849, 1851, 1853, 1855, 1857, 1859, 1861,1863, 1865, 1867, 1869, 1871, 1873, 1875, 1877, 1879, 1881, 1883, 1885,1887, 1889, 1891, 1893, 1895, 1897, 1899, 1901, 1903, 1905, 1907, 1909,1911, 1913, 1915, 1917, 1919, 1921, 1923, 1925, 1927, 1929, 1931, 1933,1935, 1937, 1939, 1941, 1943, 1945, 1947, 1949, 1951, 1953, 1955, 1957,1959, 1961, 1963, 1965, 1967, 1969, 1971, 1973, 1975, 1977, 1979, 1981,1983, 1985, 1987, 1989, 1991, 1993, 1995, 1997, 1999, 2001, 2003, 2005,2007, 2009, 2011, 2013, 2015, 2017, 2019, 2021, 2023, 2025, 2027, 2029,2031, 2033, 2035, 2037, 2039, 2041, 2043, 2045, 2047, 2049, 2051, 2053,2055, 2057, 2059, 2061, 2063, 2065, 2067, 2069, 2071, 2073, 2075, 2077,2079, 2081, 2083, 2085, 2087, 2089, 2091, 2093, 2095, 2097, 2099, 2101,2103, 2105, 2107, 2109, 2111, 2113, 2115, 2117, 2119, 2121, 2123, 2125,2127, 2129, 2131, 2133, 2135, 2137, 2139, 2141, 2143, 2145, 2147, 2149,2151, 2153, 2155, 2157, 2159, 2161, 2163, 2165, 2167, 2169, 2171, 2173,2175, 2177, 2179, 2181, 2183, 2185, 2187, 2189, 2191, 2193, 2195, 2197,2199, 2201, 2203, 2205, 2207, 2209, 2211, 2213, 2215, 2217, 2219, 2221,2223, 2225, 2227, 2229, 2231, 2233, 2235, 2237, 2239, 2241, 2243, 2245,2247, 2249, 2251, 2253, 2255, 2257, 2259, 2261, 2263, 2265, 2267, 2269,2271, 2273, 2275, 2277, 2279, 2281, 2283, 2285, 2287, 2289, 2291, 2293,2295, 2297, 2299, 2301, 2303, 2305, 2307, 2309, 2311, 2313, 2315, 2317,2319, 2321, 2323, 2325, 2327, 2329, 2331, 2333, 2335, 2337, 2339, 2341,2343, 2345, 2347, 2349, 2351, 2353, 2355, 2357, 2359, 2361, 2363, 2365,2367, 2369, 2371, 2373, 2375, 2377, 2379, 2381, 2383, 2385, 2387, 2389,2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2413,2415, 2417, 2419, 2421, 2423, 2425, 2427, 2429, 2431, 2433, 2435, 2437,2439, 2441, 2443, 2445, 2447, 2449, 2451, 2453, 2455, 2457, 2459, 2461,2463, 2465, 2467, 2469, 2471, 2473, 2475, 2477, 2479, 2481, 2483, 2485,2487, 2489, 2491, 2493, 2495, 2497, 2499, 2501, 2503, 2505, 2507, 2509,2511, 2513, 2515, 2517, 2519, 2521, 2523, 2525, 2527, 2529, 2531, 2533,2635, 2537, 2539, 2541, 2543, 2545, 2547, 2549, 2551, 2553, 2555, 2557,2559, 2561, 2563, 2565, 2567, 2569, 2571, 2573, 2575, 2577, 2579, 2581,2583, 2585, 2587, 2589, 2591, 2593, 2595, 2597, 2599, 2601, 2603, 2605,2607, 2609, 2611, 2613, 2615, 2617, 2619, 2621, 2623, 2625, 2627, 2629,2631, 2633, 2635, 2637, 2639, 2641, 2643, 2645, 2647, 2649, 2651, 2653,2655, 2657, 2659, 2661, 2663, 2665, 2667, 2669, 2671, 2673, 2675, 2677,2679, 2681, 2683, 2685, 2687, 2689, 2691, 2693, 2695, 2697, 2699, 2701,2703, 2705, 2707, 2709, 2711, 2713, 2715, 2717, 2719, 2721, 2723, 2725,2727, 2729, 2731, 2733, 2735, 2737, 2739, 2741, 2743, 2745, 2747, 2749,2751, 2753, 2755, 2757, 2759, 2761, 2763, 2765, 2767, 2769, 2771, 2773,2775, 2777, 2779, 2781, 2783, 2785, 2787, 2789, 2791, 2793, 2795, 2797,2799, 2801, 2803, 2805, 2807, 2809, 2811, 2813, 2815, 2817, 2819, 2821,2823, 2825, 2827, 2829, 2831, 2833, 2835, 2837, 2839, 2841, 2843, 2845,2847, and complementary sequences thereof, wherein the encodedpolypeptide has an amino acid sequence comprising one or more amino acidresidues selected from the group consisting of V at position 5, A atposition 27, V at position 45, Q at position 46, I at position 54, V atposition 60, V at position 65, N at position 67, H at position 72, I atposition 77, S at position 87, R at position 91, T at position 93, A atposition 95, E at position 96, M or Q at position 100, T at position103, R or T at position 117, S at position 118, R or I or L or Y atposition 121, S at position 135, R at position 139+E at position 176 orD at position 139+R at position 176, A at position 146, N at position153, H at position 166, G or A at position 177, M or C at position 178,N or D at position 179, D or T at position 180, K at position 181, N atposition 182, V at position 190, A at position 199, W or Y at position201, Y at position 205, L at position 236, T or R at position 238, F atposition 239, V at position 246, E or A or S at position 251, and P atposition 252, wherein amino acid residue position is determined byoptimal alignment with a reference amino acid sequence which is an aminoacid sequence that is encoded by the reference nucleic acid sequence.

In a further embodiment, HHDH polypeptides of the present invention havethe sequence of SEQ ID NO: 2, with one or more substitutions selectedfrom the group consisting of 15V, T27A, A45V, E46Q, M54I, A60V, A65V,T67N, Q72H, V771, Q87S, K91R, A93T, E95A, D96E, A100M/Q, A103T, S117R/T,Q118S, K121R/I/L/Y, P135S, W139R+N176E or W139D+N176R, T146A, C153N,Y166H, Y177G/A, L178M/C, H179N/D, S180D/T, E181K, D182N, E190V, V199A,H201W/Y, V205Y, V236L, W238T/R, L239F, I246V, G251E/A/S, and M252P.

In a specific embodiment, the present invention is directed to anisolated or recombinant polypeptide having HHDH activity, wherein thepolypeptide has an amino acid sequence selected from the groupconsisting of: (a) an amino acid sequence that is at least 86% identicalto a reference sequence selected from the group consisting of SEQ ID NO:750 and SEQ ID NO: 2848; (b) an amino acid sequence that is at least 93%identical to a reference sequence selected from the group consisting ofSEQ ID NO: 200 and SEQ ID NO: 1978; (c) an amino acid sequence that isat least 94% identical to reference sequence SEQ ID NO: 2496; (d) anamino acid sequence that is at least 95% identical to a referencesequence selected from the group consisting of SEQ ID NO: 264, SEQ IDNO: 266, SEQ ID NO: 470, SEQ ID NO: 476, SEQ ID NO: 2846, SEQ ID NO:2130, SEQ ID NO: 1926, SEQ ID NO: 2348, SEQ ID NO: 1984, and SEQ ID NO:2446; (e) an amino acid sequence that is at least 96% identical to areference sequence selected from the group consisting of SEQ ID NO: 116,SEQ ID NO: 448, SEQ ID NO: 2834, SEQ ID NO: 1820, SEQ ID NO: 2040, andSEQ ID NO: 2838; (f) an amino acid sequence that is at least 97%identical to a reference sequence selected from the group consisting ofSEQ ID NO: 110, SEQ ID NO: 162, SEQ ID NO: 262, SEQ ID NO: 422, SEQ IDNO: 440, SEQ ID NO: 520, SEQ ID NO: 1276, SEQ ID NO: 1710, SEQ ID NO:1824, SEQ ID NO: 2582, SEQ ID NO: 1818, SEQ ID NO: 1164, SEQ ID NO:1208, SEQ ID NO: 1750, and SEQ ID NO: 2808; (g) an amino acid sequencethat is at least 98% identical to a reference sequence selected from thegroup consisting of SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 68, SEQ IDNO: 118, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 180, SEQ ID NO:1826, SEQ ID NO: 2598, SEQ ID NO: 1324, SEQ ID NO: 1898, SEQ ID NO:2146, SEQ ID NO: 1762, SEQ ID NO: 2580, SEQ ID NO: 2724, SEQ ID NO:1698, SEQ ID NO:1648, SEQ ID NO: 2576, SEQ ID NO: 2790, and SEQ ID NO:1760; and (h) an amino acid sequence that is at least 99% identical to areference sequence selected from the group consisting of SEQ ID NO: 4,SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 34, SEQ ID NO:38, SEQ ID NO: 44, SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 66, SEQ IDNO: 80, SEQ ID NO: 84, SEQ ID NO: 114, SEQ ID NO: 154, SEQ ID NO: 158,SEQ ID NO: 170, SEQ ID NO: 264, SEQ ID NO: 266, SEQ ID NO: 270, SEQ IDNO: 470, SEQ ID NO: 476, SEQ ID NO: 1858, SEQ ID NO: 1212, SEQ ID NO:2566, SEQ ID NO: 1388, SEQ ID NO: 1316, SEQ ID NO: 1058, SEQ ID NO:1506, SEQ ID NO: 1266, SEQ ID NO: 1616, SEQ ID NO: 1292, SEQ ID NO:1770, SEQ ID NO: 1758, SEQ ID NO: 1656, SEQ ID NO: 1800, SEQ ID NO:1200, SEQ ID NO: 1354, SEQ ID NO: 1248, SEQ ID NO: 2594, SEQ ID NO:1508, SEQ ID NO: 2680, SEQ ID NO: 1904, SEQ ID NO: 1364, SEQ ID NO:1580, SEQ ID NO: 2214, SEQ ID NO: 2710, SEQ ID NO: 1670, SEQ ID NO:1286, SEQ ID NO: 2784, SEQ ID NO: 2782, SEQ ID NO: 1174, SEQ ID NO:1618, SEQ ID NO: 970, SEQ ID NO: 1214, SEQ ID NO: 1780, SEQ ID NO: 1452,SEQ ID NO: 1766, and SEQ ID NO: 1156.

In another aspect, the present invention is directed to an isolated orrecombinant HHDH polypeptide having an amino acid sequence that has asubstitution, deletion, and/or insertion of from one to twenty aminoacid residues in a sequence selected from the group consisting of SEQ IDNO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 34, 36, 38,40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74,76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136,138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164,166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192,194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220,222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248,250, 252, 254, 256, 258. 260, 262, 264, 266, 268, 270, 272, 274, 276,278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304,306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332,334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360,362, 364, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390,392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418,420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446,448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474,476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502,504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530,532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558,560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586,588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614,616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642,644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670,672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698,700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726,728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754,756, 758, 760, 762, 764, 766, 768, 770, 772, 774, 776, 778, 780, 782,784, 786, 788, 790, 792, 794, 796, 798, 800, 802, 804, 806, 808, 810,812, 814, 816, 818, 820, 822, 824, 826, 828, 830, 832, 834, 836, 838,840, 842, 844, 846, 848, 850, 852, 854, 856, 858, 860, 862, 864, 866,868, 870, 872, 874, 876, 878, 880, 882, 884, 886, 888, 890, 892, 894,896, 898, 900, 902, 904, 906, 908, 910, 912, 914, 916, 918, 920, 922,924, 926, 928, 930, 932, 934, 936, 938, 940, 942, 944, 946, 948, 950,952, 954, 956, 958, 960, 962, 964, 966, 968, 970, 972, 974, 976, 978,980, 982, 984, 986, 988, 990, 992, 994, 996, 998, 1000, 1002, 1004,1006, 1008, 1010, 1012, 1014, 1016, 1018, 1020, 1022, 1024, 1026, 1028,1030, 1032, 1034, 1036, 1038, 1040, 1042, 1044, 1046, 1048, 1050, 1052,1054, 1056, 1058, 1060, 1062, 1064, 1066, 1068, 1070, 1072, 1074, 1076,1078, 1080, 1082, 1084, 1086, 1088, 1090, 1092, 1094, 1096, 1098, 1100,1102, 1104, 1106, 1108, 1110, 1112, 1114, 1116, 1118, 1120, 1122, 1124,1126, 1128, 1130, 1132, 1134, 1136, 1138, 1140, 1142, 1144, 1146, 1148,1150, 1152, 1154, 1156, 1158, 1160, 1162, 1164, 1166, 1168, 1170, 1172,1174, 1176, 1178, 1180, 1182, 1184, 1186, 1188, 1190, 1192, 1194, 1196,1198, 1200, 1202, 1204, 1206, 1208, 1210, 1212, 1214, 1216, 1218, 1220,1222, 1224, 1226, 1228, 1230, 1232, 1234, 1236, 1238, 1240, 1242, 1244,1246, 1248, 1250, 1252, 1254, 1256, 1258, 1260, 1262, 1264, 1266, 1268,1270, 1272, 1274, 1276, 1278, 1280, 1282, 1284, 1286, 1288, 1290, 1292,1294, 1296, 1298, 1300, 1302, 1304, 1306, 1308, 1310, 1312, 1314, 1316,1318, 1320, 1322, 1324, 1326, 1328, 1330, 1332, 1334, 1336, 1338, 1340,1342, 1344, 1346, 1348, 1350, 1352, 1354, 1356, 1358, 1360, 1362, 1364,1366, 1368, 1370, 1372, 1374, 1376, 1378, 1380, 1382, 1384, 1386, 1388,1390, 1392, 1394, 1396, 1398, 1400, 1402, 1404, 1406, 1408, 1410, 1412,1414, 1416, 1418, 1420, 1422, 1424, 1426, 1428, 1430, 1432, 1434, 1436,1438, 1440, 1442, 1444, 1446, 1448, 1450, 1452, 1454, 1456, 1458, 1460,1462, 1464, 1466, 1468, 1470, 1472, 1474, 1476, 1478, 1480, 1482, 1484,1486, 1488, 1490, 1492, 1494, 1496, 1498, 1500, 1502, 1504, 1506, 1508,1510, 1512, 1514, 1516, 1518, 1520, 1522, 1524, 1526, 1528, 1530, 1532,1534, 1536, 1538, 1540, 1542, 1544, 1546, 1548, 1550, 1552, 1554, 1556,1558, 1560, 1562, 1564, 1566, 1568, 1570, 1572, 1574, 1576, 1578, 1580,1582, 1584, 1586, 1588, 1590, 1592, 1594, 1596, 1598, 1600, 1602, 1604,1606, 1608, 1610, 1612, 1614, 1616, 1618, 1620, 1622, 1624, 1626, 1628,1630, 1632, 1634, 1636, 1638, 1640, 1642, 1644, 1646, 1648, 1650, 1652,1654, 1656, 1658, 1660, 1662, 1664, 1666, 1668, 1670, 1672, 1674, 1676,1678, 1680, 1682, 1684, 1686, 1688, 1690, 1692, 1694, 1696, 1698, 1700,1702, 1704, 1706, 1708, 1710, 1720, 1722, 1724, 1726, 1728, 1730, 1732,1734, 1736, 1738, 1740, 1742, 1744, 1746, 1748, 1750, 1752, 1754, 1756,1758, 1760, 1762, 1764, 1766, 1768, 1770, 1772, 1774, 1776, 1778, 1780,1782, 1784, 1786, 1788, 1790, 1792, 1794, 1796, 1798, 1800, 1802, 1804,1806, 1808, 1810, 1812, 1814, 1816, 1818, 1820, 1822, 1824, 1826, 1828,1830, 1832, 1834, 1836, 1838, 1840, 1842, 1844, 1846, 1848, 1850, 1852,1854, 1856, 1858, 1860, 1862, 1864, 1866, 1868, 1870, 1872, 1874, 1876,1878, 1880, 1882, 1884, 1886, 1888, 1890, 1892, 1894, 1896, 1898, 1900,1902, 1904, 1906, 1908, 1910, 1912, 1914, 1916, 1918, 1920, 1922, 1924,1926, 1928, 1930, 1932, 1934, 1936, 1938, 1940, 1942, 1944, 1946, 1948,1950, 1952, 1954 1956, 1958, 1960, 1962, 1964, 1966, 1968, 1970, 1972,1974, 1976, 1978, 1980, 1982, 1984, 1986, 1988, 1990, 1992, 1994, 1996,1998, 2000, 2002, 2004, 2006, 2008, 2010, 2012, 2014, 2016, 2018, 2020,2022, 2024, 2026, 2028, 2030, 2032, 2034, 2036, 2038, 2040, 2042, 2044,2046, 2048, 2050, 2052, 2054, 2056, 2058, 2060, 2062, 2064, 2066, 2068,2070, 2072, 2074, 2076, 2078, 2080, 2082, 2084, 2086, 2088, 2090, 2092,2094, 2096, 2098, 2100, 2102, 2104, 2106, 2108, 2110, 2112, 2114, 2116,2118, 2120, 2122, 2124, 2126, 2128, 2130, 2132, 2134, 2136, 2138, 2140,2142, 2144, 2146, 2148, 2150, 2152, 2154, 2156, 2158, 2160, 2162, 2164,2166, 2168, 2170, 2172, 2174, 2176, 2178, 2180, 2182, 2184, 2186, 2188,2190, 2192, 2194, 2196, 2198, 2200, 2202, 2204, 2206, 2208, 2210, 2212,2214, 2216, 2218, 2220, 2222, 2224, 2226, 2228, 2230, 2232, 2234, 2236,2238, 2240, 2242, 2244, 2246, 2248, 2250, 2252, 2254, 2256, 2258, 2260,2262, 2264, 2266, 2268, 2270, 2272, 2274, 2276, 2278, 2280, 2282, 2284,2286, 2288, 2300, 2302, 2304, 2306, 2308, 2310, 2312, 2314, 2316, 2318,2320, 2322, 2324, 2326, 2328, 2330, 2332, 2334, 2336, 2338, 2340, 2342,2344, 2346, 2348, 2350, 2352, 2354, 2356, 2358, 2360, 2362, 2364, 2366,2368, 2370, 2372, 2374, 2376, 2378, 2380, 2382, 2384, 2386, 2388, 2390,2392, 2394, 2396, 2398, 2400, 2402, 2404, 2406, 2408, 2410, 2412, 2414,2416, 2418, 2420, 2422, 2424, 2426, 2428, 2430, 2432, 2434, 2436, 2438,2440, 2442, 2444, 2446, 2448, 2450, 2452, 2454, 2456, 2458, 2460, 2462,2464, 2466, 2468, 2470, 2472, 2474, 2476, 2478, 2480, 2482, 2484, 2486,2488, 2490, 2492, 2494, 2496, 2498, 2500, 2502, 2504, 2506, 2508, 2510,2512, 2514, 2516, 2518, 2520, 2522, 2524, 2526, 2528, 2530, 2532, 2534,2536, 2538, 2540, 2542, 2544, 2546, 2548, 2550, 2552, 2554, 2556, 2558,2560, 2562, 2564, 2566, 2568, 2570, 2572, 2574, 2576, 2578, 2580, 2582,2584, 2586, 2588, 2590, 2592, 2594, 2596, 2598, 2600, 2602, 2604, 2606,2608, 2610, 2612, 2614, 2616, 2618, 2620, 2622, 2624, 2626, 2628, 2630,2632, 2634, 2636, 2638, 2640, 2642, 2644, 2646, 2648, 2650, 2652, 2654,2656, 2658, 2660, 2662, 2664, 2666, 2668, 2670, 2672, 2674, 2676, 2678,2680, 2682, 2684, 2686, 2688, 2690, 2700, 2702, 2704, 2706, 2708, 2710,2712, 2714, 2716, 2718, 2720, 2722, 2724, 2726, 2728, 2730, 2732, 2734,2736, 2738, 2740, 2742, 2744, 2746, 2748, 2750, 2752, 2754, 2756, 2758,2760, 2762, 2764, 2766, 2768, 2770, 2772, 2774, 2776, 2778, 2780, 2782,2784, 2786, 2788, 2790, 2792, 2794, 2796, 2798, 2800, 2802, 2804, 2806,2808, 2810, 2812, 2814, 2816, 2818, 2820, 2822, 2824, 2826, 2828, 2830,2832, 2834, 2836, 2838, 2840, 2842, 2844, 2846, and 2848, and whereinthe encoded HHDH polypeptide has HHDH activity that is greater than theHHDH activity of wildtype HHDH (SEQ ID NO: 2). Typically, the HHDHactivity is at least 1.4 fold greater than the HHDH polypeptide of SEQID NO: 2, as measured in the assay of Example 5A; or at least 1.5-foldgreater than the HHDH polypeptide of SEQ ID NO: 730, as measured in theassay of Example 5D.

In a further aspect, the present invention is directed to HHDHpolypeptides having improved HHDH enzymatic activity relative to thewild-type HHDH of SEQ ID NO: 2, as measured in the assay described inExample 5A.

In another aspect, the present invention is directed to HHDHpolypeptides having greater activity than the improved HHDH polypeptideof SEQ ID NO: 730 under the commercially relevant conditions describedin Example 5D.

In another aspect, the present invention provides HHDH polypeptideshaving the combination of at least one of the novel substitutionsdescribed herein, coupled with one or more wildtype amino acid residuesselected from D at position 79, P at position 175, and Y at position187.

In a still further aspect, the present invention provides isolated orrecombinant polynucleotides that encode the HHDH polypeptides of thepresent invention.

The present invention is also directed to a nucleic acid constructcomprising a promoter that is operably linked to a polynucleotide of thepresent invention, as well as host cells, and methods of producing andusing the HHDH polypeptides.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a 3944 bp expression vector (PCK110700) of the presentinvention comprising a p15A origin of replication (P15A ori), a lacIrepressor, a T5 promoter, a T7 ribosomal binding site (T7g10), and achloramphenicol resistance gene (camR).

FIG. 2 depicts the percent conversion vs. time for the reactions ofethyl (S)-4-chloro-3-hydroxybutyrate with aqueous hydrocyanic acid inthe presence of various halohydrin dehalogenase enzymes that aredescribed in Examples 8 through 12.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING SUBMITTED ON COMPACT DISC

The Sequence Listing submitted concurrently herewith is provided onduplicate (2) copies of a compact disc, each of which contains the file,“16028US05-0353.510US.txt” (size on disk is 8,585,216 bytes and the dateof creation is Oct. 28, 2005). Copy 1 and Copy 2 are identical. TheSequence Listing on the compact discs is hereby incorporated byreference in its entirety.

DETAILED DESCRIPTION

HHDH Polypeptides

The present invention provides novel polypeptides having halohydrindehalogenase (“HHDH”) activity, as well as the polynucleotides thatencode them. HHDH polypeptides of the present invention are suitable forcatalyzing the conversion of 4-halo-3-hydroxybutyric acid derivatives to4-substituted-3-hydroxybutyric acid derivatives using, for example, themethods described in the patent application entitled, “EnzymaticProcesses for the Production of 4-Substituted-3-Hydroxybutyric AcidDerivatives,” filed on Aug. 11, 2003 and assigned U.S. Ser. No.10/639,159, and International patent publication WO 2004/015132, both ofwhich are hereby incorporated herein by reference. These inventionpolypeptides are also suitable for catalyzing the conversion of vicinalhalo, hydroxy substituted carboxylic acid esters to vicinal cyano,hydroxy substituted carboxylic acid esters using, for example. themethods described in the patent application entitled, “EnzymaticProcesses for the Production of 4-Substituted-3-Hydroxybutyric AcidDerivatives and Vicinal Cyano, Hydroxy Substituted Carboxylic AcidEsters,” filed on Feb. 18, 2004 and assigned U.S. Ser. No. 10/782,258and International patent publication WO 2005/018579, both of which ishereby incorporated by reference. Polypeptides of the present inventionare particularly useful as catalysts for converting halohydrins tocyanohydrins, which are useful as pharmaceutical intermediates. In aspecific application, HHDH polypeptides of the present invention areused to catalyze the conversion of ethyl (S) 4-chloro-3-hydroxybutyrate(“ECHB”) to ethyl (R) 4-cyano-3-hydroxybutyrate (“HN”). Examplesillustrating such conversion are provided hereinbelow. A more detaileddescription of such uses is provided in the aforementioned patentapplications entitled, “Enzymatic Processes for the Production of4-Substituted-3-Hydroxybutyric Acid Derivatives” and “EnzymaticProcesses for the Production of 4-Substituted-3-Hydroxybutyric AcidDerivatives and Vicinal Cyano, Hydroxy Substituted Carboxylic AcidEsters.” Id.

The present invention has multiple aspects. In one embodiment, thepresent invention provides an isolated or recombinant HHDH polypeptidecomprising an amino acid sequence that is at least 80% identical to areference sequence selected from the group consisting of SEQ ID NOs: 2,4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76,78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136,138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164,166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192,194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220,222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248,250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276,278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304,306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332,334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360,362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388,390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416,418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444,446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472,474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500,502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528,530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556,558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584,586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612,614, 616, 618, 619, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638,640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666,668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694,696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722,724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750,752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772, 774, 776, 778,780, 782, 784, 786, 788, 790, 792, 794, 796, 798, 800, 802, 804, 806,808, 810, 812, 814, 816, 818, 820, 822, 824, 826, 828, 830, 832, 834,836, 838, 840, 842, 844, 846, 848, 850, 852, 854, 856, 858, 860, 862,864, 866, 868, 870, 872, 874, 876, 878, 880, 882, 884, 886, 888, 890,892, 894, 896, 898, 900, 902, 904, 906, 908, 910, 912, 914, 916, 918,920, 922, 924, 926, 928, 930, 932, 934, 936, 938, 940, 942, 944, 946,948, 950, 952, 954, 956, 958, 960, 962, 964, 966, 968, 970, 972, 974,976, 978, 980, 982, 984, 986, 988, 990, 992, 994, 996, 998, 1000, 1002,1004, 1006, 1008, 1010, 1012, 1014, 1016, 1018, 1020, 1022, 1024, 1026,1028, 1030, 1032, 1034, 1036, 1038, 1040, 1042, 1044, 1046, 1048, 1050,1052, 1054, 1056, 1058, 1060, 1062, 1064, 1066, 1068, 1070, 1072, 1074,1076, 1078, 1080, 1082, 1084, 1086, 1088, 1090, 1092, 1094, 1096, 1098,1100, 1102, 1104, 1106, 1108, 1110, 1112, 1114, 1116, 1118, 1120, 1122,1124, 1126, 1128, 1130, 1132, 1134, 1136, 1138, 1140, 1142, 1144, 1146,1148, 1150, 1152, 1154, 1156, 1158, 1160, 1162, 1164, 1166, 1168, 1170,1172, 1174, 1176, 1178, 1180, 1182, 1184, 1186, 1188, 1190, 1192, 1194,1196, 1198, 1110, 1112, 1114, 1116, 1118, 1120, 1122, 1124, 1126, 1128,1130, 1132, 1134, 1136, 1138, 1140, 1142, 1144, 1146, 1148, 1150, 1152,1154, 1156, 1158, 1160, 1162, 1164, 1166, 1168, 1170, 1172, 1174, 1176,1178, 1180, 1182, 1184, 1186, 1188, 1190, 1192, 1194, 1196, 1198, 1200,1202, 1204, 1206, 1208, 1210, 1212, 1214, 1216, 1218, 1220, 1222, 1224,1226, 1228, 1230, 1232, 1234, 1236, 1238, 1240, 1242, 1244, 1246, 1248,1250, 1252, 1254, 1256, 1258, 1260, 1262, 1264, 1266, 1268, 1270, 1272,1274, 1276, 1278, 1280, 1282, 1284, 1286, 1288, 1290, 1292, 1294, 1296,1298, 1300, 1302, 1304, 1306, 1308, 1310, 1312, 1314, 1316, 1318, 1320,1322, 1324, 1326, 1328, 1330, 1332, 1334, 1336, 1338, 1340, 1342, 1344,1346, 1348, 1350, 1352, 1354, 1356. 1358. 1360, 1362, 1364, 1366, 1368,1370, 1372, 1374, 1376, 1378, 1380, 1382, 1384, 1386, 1388, 1390, 1392,1394, 1396, 1398, 1400, 1402, 1404, 1406, 1408, 1410, 1412, 1414, 1416,1418, 1420, 1422, 1424, 1426, 1428, 1430, 1432, 1434, 1436, 1438, 1440,1442, 1444, 1446, 1448, 1450, 1452, 1454, 1456, 1458, 1460, 1462, 1464,1468, 1470, 1472, 1474, 1476, 1478, 1480, 1482, 1484, 1486, 1488, 1490,1492, 1494, 1496, 1498, 1500, 1502, 1504, 1506, 1508, 1510, 1512, 1514,1516, 1518, 1520, 1522, 1524, 1526, 1528, 1530, 1532, 1534, 1536, 1538,1540, 1542, 1544, 1546, 1548, 1550, 1552, 1554, 1556, 1558, 1560, 1562,1564, 1566, 1568, 1570, 1572, 1574, 1576, 1578, 1580, 1582, 1584, 1586,1588, 1590, 1592, 1594, 1596, 1598, 1600, 1602, 1604, 1606, 1608, 1610,1612, 1614, 1616, 1618, 1620, 1622, 1624, 1626, 1628, 1630, 1632, 1634,1636, 1638, 1640, 1642, 1644, 1646, 1648, 1650, 1652, 1654, 1656, 1658,1660, 1662, 1664, 1666, 1668, 1670, 1672, 1674, 1676, 1678, 1680, 1682,1684, 1684, 1686, 1688, 1690, 1692, 1694, 1696, 1698, 1700, 1702, 1704,1706, 1708, 1710, 1712, 1714, 1716, 1718, 1720, 1722, 1724, 1726, 1728,1730, 1732, 1734, 1736, 1738, 1740, 1742, 1744, 1746, 1748, 1750, 1752,1754, 1756, 1758, 1760, 1762, 1764, 1766, 1768, 1770, 1772, 1774, 1776,1778, 1780, 1782, 1784, 1786, 1788, 1790, 1792, 1794, 1796, 1798, 1800,1802, 1804, 1806, 1808, 1810, 1812, 1814, 1816, 1818, 1820, 1822, 1824,1826, 1828, 1830, 1832, 1834, 1836, 1838, 1840, 1842, 1844, 1846, 1848,1850, 1852, 1854, 1856, 1858, 1860, 1862, 1864, 1866, 1868, 1870, 1872,1874, 1876, 1878, 1880, 1882, 1884, 1886, 1888, 1890, 1892, 1894, 1896,1898, 1900, 1902, 1904, 1906, 1908, 1910, 1912, 1914, 1916, 1918, 1920,1922, 1924, 1926, 1928, 1930, 1932, 1934, 1936, 1938, 1940, 1942, 1944,1946, 1948, 1950, 1952, 1954, 1956, 1958, 1960, 1962, 1964, 1966, 1968,1970, 1972, 1974, 1976, 1978, 1980, 1982, 1984, 1986, 1988, 1990, 1992,1994, 1996, 1998, 2000, 2002, 2004, 2006, 2008, 2010, 2012, 2014, 2016,2018, 2020, 2022, 2024, 2026, 2028, 2030, 2032, 2034, 2036, 2038, 2040,2042, 2044, 2046, 2048, 2050, 2052, 2054, 2056, 2058, 2060, 2062, 2064,2066, 2068, 2070, 2072, 2074, 2076, 2078, 2080, 2082, 2084, 2086, 2088,2090, 2092, 2094, 2096, 2098, 2100, 2102, 2104, 2106, 2108, 2110, 2112,2114, 2116, 2118, 2120, 2122, 2124, 2126, 2128, 2130, 2132, 2134, 2136,2138, 2140, 2142, 2144, 2146, 2148, 2150, 2152, 2154, 2156, 2158, 2160,2162, 2164, 2166, 2168, 2170, 2172, 2174, 2176, 2178, 2180, 2182, 2184,2186, 2188, 2190, 2192, 2194, 2196, 2198, 2200, 2202, 2204, 2206, 2208,2210, 2212, 2214, 2216, 2218, 2220, 2222, 2224, 2226, 2228, 2230, 2232,2234, 2236, 2238, 2240, 2242, 2244, 2246, 2248, 2250, 2252, 2254, 2256,2258, 2260, 2262, 2264, 2266, 2268, 2270, 2272, 2274, 2276, 2278, 2280,2282, 2284, 2286, 2288, 2290, 2292, 2294, 2296, 2298, 2300, 2302, 2304,2306, 2308, 2310, 2312, 2314, 2316, 2318, 2320, 2322, 2324, 2326, 2328,2330, 2332, 2334, 2336, 2338, 2340, 2342, 2344, 2346, 2348, 2350, 2352,2354, 2356, 2358, 2360, 2362, 2364, 2366, 2368, 2370, 2372, 2374, 2376,2378, 2380, 2382, 2384, 2386, 2388, 2390, 2392, 2394, 2396, 2398, 2400,2402, 2404, 2406, 2408, 2410, 2412, 2414, 2416, 2418, 2420, 2422, 2424,2426, 2428, 2430, 2432, 2434, 2436, 2438, 2440, 2442, 2444, 2446, 2448,2450, 2452, 2454, 2456, 2458, 2460, 2462, 2464, 2466, 2468, 2470, 2472,2474, 2476, 2478, 2480, 2482, 2484, 2486, 2488, 2490, 2492, 2494, 2496,2498, 2500, 2502, 2504, 2506, 2508, 2510, 2512, 2514, 2516, 2518, 2520,2522, 2524, 2526, 2528, 2530, 2532, 2534, 2526, 2528, 2530, 2532, 2534,2536, 2538, 2540, 2542, 2544, 2546, 2548, 2550, 2552, 2554, 2556, 2558,2560, 2562, 2564, 2566, 2568, 2570, 2572, 2574, 2576, 2578, 2580, 2582,2584, 2586, 2588, 2590, 2592, 2594, 2596, 2598, 2600, 2602, 2604, 2606,2608, 2610, 2612, 2614, 2616, 2618, 2620, 2622, 2624, 2626, 2628, 2630,2632, 2634, 2636, 2638, 2640, 2642, 2644, 2646, 2648, 2650, 2652, 2654,2656, 2658, 2660, 2662, 2664, 2668, 2670, 2672, 2674, 2676, 2678, 2680,2682, 2684, 2686, 2688, 2690, 2692, 2694, 2696, 2698, 2700, 2702, 2704,2706, 2708, 2710, 2712, 2714, 2716, 2718, 2720, 2722, 2724, 2726, 2728,2730, 2732, 2734, 2736, 2738, 2740, 2742, 2744, 2746, 2748, 2750, 2752,2754, 2756, 2758, 2760, 2762, 2764, 2766, 2768, 2770, 2772, 2774, 2776,2778, 2780, 2782, 2784, 2786, 2788, 2790, 2792, 2794, 2796, 2798, 2800,2802, 2804, 2806, 2808, 2810, 2812, 2814, 2816, 2818, 2820, 2822, 2824,2826, 2828, 2830, 2832, 2834, 2836, 2838, 2840, 2842, 2844, 2846, and2848, wherein the amino acid sequence further comprises one or moreamino acid residues selected from the group consisting of V at position5, A at position 27, V at position 45, Q at position 46, I at position54, V at position 60, V at position 65, N at position 67, H at position72, I at position 77, S at position 87, R at position 91, T at position93, A at position 95, E at position 96, M or Q at position 100, T atposition 103, R or T at position 117, S at position 118, R or I or L orY at position 121, S at position 135, R at position 139+E at position176 or D at position 139+R at position 176, A at position 146, N atposition 153, H at position 166, G or A at position 177, M or C atposition 178, N or D at position 179, D or T at position 180, K atposition 181, N at position 182, V at position 190, A at position 199, Wor Y at position 201, Y at position 205, L at position 236, T or R atposition 238, F at position 239, V at position 246, E or A or S atposition 251, and P at position 252, wherein amino acid residue positionis determined by optimal alignment of the amino acid sequence of theHHDH polypeptide with the reference sequence. Sometimes, these HHDHpolypeptides have an amino acid sequence that is at least 81% identical,82% identical, 83% identical, 84% identical, 85% identical, 86%identical, 87% identical, 88% identical, 89% identical, 90% identical,91% identical 92% identical, 93% identical, 94% identical, 95%,identical, 96% identical, 97% identical, 98% or 99% identical to thereference sequence.

Typically, the reference sequence is selected from the group consistingof SEQ ID NO: 2, SEQ ID NO: 200, SEQ ID NO: 730, SEQ ID NO: 750, SEQ IDNO: 1926, SEQ ID NO: 1984, SEQ ID NO: 2040, SEQ ID NO: 2130, SEQ ID NO:2348, and SEQ ID NO: 2848.

In some embodiments, the above-described HHDH polypeptides are encodedby an amino acid sequence that comprises one or more amino acid residuesselected from the subgroup consisting of A at position 27, Q at position46, I at position 54, V at position 60, V at position 65, S at position87, R at position 91, A at position 95, E at position 96, M or Q atposition 100, R at position 117, S at position 118, R at position 121, Sat position 135, A at position 146, N at position 153, M at position178, N at position 179, N at position 182, V at position 190, A atposition 199, Y at position 201, Y at position 205, L at position 236, Tat position 238, and E or A at position 251.

In certain embodiments, these HHDH polypeptides are encoded by an aminoacid sequence having one or more amino acid residues selected from thesubgroup consisting of V at position 5, V at position 45, N at position67, H at position 72, I at position 77, T at position 93, T at position103, T at position 117, S at position 118, I or L or Y at position 121,R at position 139+E at position 176 or D at position 139+R at position176, H at position 166, G or A at position 177, C at position 178, D atposition 179, W at position 201, R at position 238, F at position 239, Vat position 246, S at position 251, and P at position 252. In otherembodiments, these HHDH polypeptides are encoded by an amino acidsequence that comprises one or more residues selected from the groupconsisting of I at position 54, V at position 60, N at position 67, H atposition 72, T at position 93, M at position 100, T at position 117, Rat position 121, S at position 135, A at position 146, A or G atposition 177, M or C at position 178, D at position 179, W at position201, Y at position 205, R at position 238, F at position 239, and V atposition 246. Some of these HHDH polypeptides have an amino acidsequence that comprises one or more amino acid residues selected fromthe subgroup group consisting of N at position 67, H at position 72, Tat position 117, R at position 121, S at position 135, A at position146, A at position 177, D at position 179, W at position 201, Y atposition 205, and V at position 246. HHDH polypeptides of the presentinvention include those having an amino acid sequence comprising thefollowing combination of amino acid residues: N at position 67+H atposition 72+T at position 117+R at position 121+S at position 135+A atposition 146+A at position 154+A at position 177+D at position 179+W atposition 201+Y at position 205+V at position 246. In other embodiments,the HHDH polypeptides have an amino acid sequence that comprises one ormore amino acid residues selected from the group consisting of I atposition 54, V at position 60, E at position 91, T at position 93, M atposition 100, G at position 177, M or C at position 178, R at position238, and F at position 239.

In addition to the amino acid residues described above, the amino acidsequences of the HHDH polypeptides described in paragraphs 024-027 mayfurther comprise one or more amino acid residues selected from the groupconsisting of T at position 2, A or P or S at position 3, V at position4, D at position 6, I or F at position 9, L at position 10, S atposition 13, S at position 14, K at position 15, C at position 16, T orR at position 17, C or S or K at position 20, T at position 24, Q atposition 26, F at position 28, T at position 29, A at position 30, I atposition 31, G at position 33, R at position 34, L at position 35, N atposition 36, H at position 37, Q at position 38, D at position 40, L atposition 44, P at position 45 (if not V from paragraphs 024-027), P or Aat position 47, N at position 51, I at position 52, V at position 54 (ifnot I), R at position 55, D at position 56, K at position 58, G or D atposition 61, V at position 63, L at position 70, R at position 72 (ifnot H), I at position 75, P at position 76, C at position 78, Q atposition 80, A or Y at position 82, P at position 83, S or L or V atposition 84, A at position 85, W at position 86, R at position 87, E atposition 91 (if not R), D at position 93 (if not T), Q or G at position95 (if not A), N at position 96 (if not E), D at position 99, T atposition 100, K at position 107, A at position 112, T or S or G atposition 114, A at position 115, P at position 117 (if not R or T), N atposition 120, E at position 121 (if not R or I or L or Y), P at position122, R at position 126, V at position 130, S at position 133, A or V atposition 134, L or W or V at position 136, H at position 139 (if not Ror D), I or R at position 142, S at position 144, S at position 146 (ifnot A), T at position 152, S or A at position 154, V at position 168, Tat position 169, A at position 174, F at position 177 (if not G or A), Vat position 178 (if not M or C), I at position 180, G or I at position181, K at position 184, Y at position 186, S at position 189, L atposition 194, S at position 195, N at position 198, M at position 199(if not A), R at position 203, E at position 215, T at position 222, Gat position 236 (if not L), V at position 237, L at position 238 (if notT or R), T at position 240, I or A or V at position 245, Y at position249, V or I at position 252 (if not P), and V at position 254, whereinamino acid residue position is determined by optimal alignment of theamino acid sequence of the HHDH polypeptide with the reference sequence.

Some of the HHDH polypeptides described in paragraphs 024-027 have anamino acid sequence that further comprises one or more amino acidresidues selected from the subgroup consisting of H at position 37, Q atposition 38, I at position 52, L at position 70, I at position 75, A atposition 82, P at position 83, V at position 84, W at position 86, R atposition 87, D at position 99, T at position 100, K at position 107, Aat position 112, A at position 134, A at position 154, A at position174, V at position 178 (if not M or C), G at position 181, Y at position186, S at position 189, S at position 195, R at position 203, T atposition 222, and V at position 245.

Some of the HHDH polypeptides described in paragraphs 024-027 furthercomprise one or more amino acid residues selected from one of thefollowing subgroups: (1) T at position 2, A or P or S at position 3, Vat position 4, D at position 6, I or F at position 9, L at position 10,S at position 13, S at position 14, K at position 15, C at position 16,T or R at position 17, C or S or K at position 20, T at position 24, Qat position 26, F at position 28, T at position 29, A at position 30, Iat position 31, G at position 33, R at position 34, L at position 35, Nat position 36, H at position 37, D at position 40, L at position 44, Pat position 45 (if not I), P or A at position 47, N at position 51, V atposition 54 (if not I), R at position 55, D at position 56, K atposition 58, G or D at position 61, V at position 63, R at position 72(if not H), I at position 75, P at position 76, C at position 78, Q atposition 80, Y at position 82, S or L at position 84, A at position 85,E at position 91 (if not R), D at position 93 (if not T), Q or G atposition 95 (if not A), N at position 96 (if not E), K at position 107,A at position 112, T or S or G at position 114, A at position 115, P atposition 117 (if not R or T), N at position 120, E at position 121 (ifnot R or I or L or Y), P at position 122, R at position 126, V atposition 130, S at position 133, A or V at position 134, L or W or V atposition 136, H at position 139 (if not R or D), I or R at position 142,S at position 144, S at position 146 (if not A), T at position 152, S orA at position 154, V at position 168, T at position 169, F at position177 (if not G or A), V at position 178 (if not M or C), I at position180, G or I at position 181, K at position 184, Y at position 186, L atposition 194, N at position 198, M at position 199 (if not A), E atposition 215, G at position 236, V at position 237, L at position 238(if not T or R), T at position 240, 1 or A or V at position 245, Y atposition 249, V or I at position 252 (if not P), and V at position 254;or (2) Q at position 38, I at position 52, L at position 70, A atposition 82, P at position 83, V at position 84, W at position 86, R atposition 87, D at position 99, T at position 100, A at position 174, Sat position 189, S at position 195, R at position 203, and T at position222; or (3) H at position 37, I at position 75, K at position 107, A atposition 112, A at position 134, A at position 154, V at position 178(if not M or C), G at position 181, Y at position 186, T at position222, and V at position 245. The parentheticals in the above paragraphs(“if not X”) refers to the proviso in which the specified residue isexcluded from the specified amino acid residue position if amino acidresidue X is selected from paragraphs 024-027 for that residue position.

In one embodiment, the present invention provides an isolated orrecombinant HHDH polypeptide comprising an amino acid sequence that isat least 80% identical to a reference sequence selected from the groupconsisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96,98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152,154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180,182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208,210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236,238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264,266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292,294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320,322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348,350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376,378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404,406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432,434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460,462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488,490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516,518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544,546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572,574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600,602, 604, 606, 608, 610, 612, 614, 616, 618, 619, 620, 622, 624, 626,628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654,656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682,684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710,712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738,740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766,768, 770, 772, 774, 776, 778, 780, 782, 784, 786, 788, 790, 792, 794,796, 798, 800, 802, 804, 806, 808, 810, 812, 814, 816, 818, 820, 822,824, 826, 828, 830, 832, 834, 836, 838, 840, 842, 844, 846, 848, 850,852, 854, 856, 858, 860, 862, 864, 866, 868, 870, 872, 874, 876, 878,880, 882, 884, 886, 888, 890, 892, 894, 896, 898, 900, 902, 904, 906,908, 910, 912, 914, 916, 918, 920, 922, 924, 926, 928, 930, 932, 934,936, 938, 940, 942, 944, 946, 948, 950, 952, 954, 956, 958, 960, 962,964, 966, 968, 970, 972, 974, 976, 978, 980, 982, 984, 986, 988, 990,992, 994, 996, 998, 1000, 1002, 1004, 1006, 1008, 1010, 1012, 1014,1016, 1018, 1020, 1022, 1024, 1026, 1028, 1030, 1032, 1034, 1036, 1038,1040, 1042, 1044, 1046, 1048, 1050, 1052, 1054, 1056, 1058, 1060, 1062,1064, 1066, 1068, 1070, 1072, 1074, 1076, 1078, 1080, 1082, 1084, 1086,1088, 1090, 1092, 1094, 1096, 1098, 1100, 1102, 1104, 1106, 1108, 1110,1112, 1114, 1116, 1118, 1120, 1122, 1124, 1126, 1128, 1130, 1132, 1134,1136, 1138, 1140, 1142, 1144, 1146, 1148, 1150, 1152, 1154, 1156, 1158,1160, 1162, 1164, 1166, 1168, 1170, 1172, 1174, 1176, 1178, 1180, 1182,1184, 1186, 1188, 1190, 1192, 1194, 1196, 1198, 1110, 1112, 1114, 1116,1118, 1120, 1122, 1124, 1126, 1128, 1130, 1132, 1134, 1136, 1138, 1140,1142, 1144, 1146, 1148, 1150, 1152, 1154, 1156, 1158, 1160, 1162, 1164,1166, 1168, 1170, 1172, 1174, 1176, 1178, 1180, 1182, 1184, 1186, 1188,1190, 1192, 1194, 1196, 1198, 1200, 1202, 1204, 1206, 1208, 1210, 1212,1214, 1216, 1218, 1220, 1222, 1224, 1226, 1228, 1230, 1232, 1234, 1236,1238, 1240, 1242, 1244, 1246, 1248, 1250, 1252, 1254, 1256, 1258, 1260,1262, 1264, 1266, 1268, 1270, 1272, 1274, 1276, 1278, 1280, 1282, 1284,1286, 1288, 1290, 1292, 1294, 1296, 1298, 1300, 1302, 1304, 1306, 1308,1310, 1312, 1314, 1316, 1318, 1320, 1322, 1324, 1326, 1328, 1330, 1332,1334, 1336, 1338, 1340, 1342, 1344, 1346, 1348, 1350, 1352, 1354, 1356.1358. 1360, 1362, 1364, 1366, 1368, 1370, 1372, 1374, 1376, 1378, 1380,1382, 1384, 1386, 1388, 1390, 1392, 1394, 1396, 1398, 1400, 1402, 1404,1406, 1408, 1410, 1412, 1414, 1416, 1418, 1420, 1422, 1424, 1426, 1428,1430, 1432, 1434, 1436, 1438, 1440, 1442, 1444, 1446, 1448, 1450, 1452,1454, 1456, 1458, 1460, 1462, 1464, 1468, 1470, 1472, 1474, 1476, 1478,1480, 1482, 1484, 1486, 1488, 1490, 1492, 1494, 1496, 1498, 1500, 1502,1504, 1506, 1508, 1510, 1512, 1514, 1516, 1518, 1520, 1522, 1524, 1526,1528, 1530, 1532, 1534, 1536, 1538, 1540, 1542, 1544, 1546, 1548, 1550,1552, 1554, 1556, 1558, 1560, 1562, 1564, 1566, 1568, 1570, 1572, 1574,1576, 1578, 1580, 1582, 1584, 1586, 1588, 1590, 1592, 1594, 1596, 1598,1600, 1602, 1604, 1606, 1608, 1610, 1612, 1614, 1616, 1618, 1620, 1622,1624, 1626, 1628, 1630, 1632, 1634, 1636, 1638, 1640, 1642, 1644, 1646,1648, 1650, 1652, 1654, 1656, 1658, 1660, 1662, 1664, 1666, 1668, 1670,1672, 1674, 1676, 1678, 1680, 1682, 1684, 1684, 1686, 1688, 1690, 1692,1694, 1696, 1698, 1700, 1702, 1704, 1706, 1708, 1710, 1712, 1714, 1716,1718, 1720, 1722, 1724, 1726, 1728, 1730, 1732, 1734, 1736, 1738, 1740,1742, 1744, 1746, 1748, 1750, 1752, 1754, 1756, 1758, 1760, 1762, 1764,1766, 1768, 1770, 1772, 1774, 1776, 1778, 1780, 1782, 1784, 1786, 1788,1790, 1792, 1794, 1796, 1798, 1800, 1802, 1804, 1806, 1808, 1810, 1812,1814, 1816, 1818, 1820, 1822, 1824, 1826, 1828, 1830, 1832, 1834, 1836,1838, 1840, 1842, 1844, 1846, 1848, 1850, 1852, 1854, 1856, 1858, 1860,1862, 1864, 1866, 1868, 1870, 1872, 1874, 1876, 1878, 1880, 1882, 1884,1886, 1888, 1890, 1892, 1894, 1896, 1898, 1900, 1902, 1904, 1906, 1908,1910, 1912, 1914, 1916, 1918, 1920, 1922, 1924, 1926, 1928, 1930, 1932,1934, 1936, 1938, 1940, 1942, 1944, 1946, 1948, 1950, 1952, 1954, 1956,1958, 1960, 1962, 1964, 1966, 1968, 1970, 1972, 1974, 1976, 1978, 1980,1982, 1984, 1986, 1988, 1990, 1992, 1994, 1996, 1998, 2000, 2002, 2004,2006, 2008, 2010, 2012, 2014, 2016, 2018, 2020, 2022, 2024, 2026, 2028,2030, 2032, 2034, 2036, 2038, 2040, 2042, 2044, 2046, 2048, 2050, 2052,2054, 2056, 2058, 2060, 2062, 2064, 2066, 2068, 2070, 2072, 2074, 2076,2078, 2080, 2082, 2084, 2086, 2088, 2090, 2092, 2094, 2096, 2098, 2100,2102, 2104, 2106, 2108, 2110, 2112, 2114, 2116, 2118, 2120, 2122, 2124,2126, 2128, 2130, 2132, 2134, 2136, 2138, 2140, 2142, 2144, 2146, 2148,2150, 2152, 2154, 2156, 2158, 2160, 2162, 2164, 2166, 2168, 2170, 2172,2174, 2176, 2178, 2180, 2182, 2184, 2186, 2188, 2190, 2192, 2194, 2196,2198, 2200, 2202, 2204, 2206, 2208, 2210, 2212, 2214, 2216, 2218, 2220,2222, 2224, 2226, 2228, 2230, 2232, 2234, 2236, 2238, 2240, 2242, 2244,2246, 2248, 2250, 2252, 2254, 2256, 2258, 2260, 2262, 2264, 2266, 2268,2270, 2272, 2274, 2276, 2278, 2280, 2282, 2284, 2286, 2288, 2290, 2292,2294, 2296, 2298, 2300, 2302, 2304, 2306, 2308, 2310, 2312, 2314, 2316,2318, 2320, 2322, 2324, 2326, 2328, 2330, 2332, 2334, 2336, 2338, 2340,2342, 2344, 2346, 2348, 2350, 2352, 2354, 2356, 2358, 2360, 2362, 2364,2366, 2368, 2370, 2372, 2374, 2376, 2378, 2380, 2382, 2384, 2386, 2388,2390, 2392, 2394, 2396, 2398, 2400, 2402, 2404, 2406, 2408, 2410, 2412,2414, 2416, 2418, 2420, 2422, 2424, 2426, 2428, 2430, 2432, 2434, 2436,2438, 2440, 2442, 2444, 2446, 2448, 2450, 2452, 2454, 2456, 2458, 2460,2462, 2464, 2466, 2468, 2470, 2472, 2474, 2476, 2478, 2480, 2482, 2484,2486, 2488, 2490, 2492, 2494, 2496, 2498, 2500, 2502, 2504, 2506, 2508,2510, 2512, 2514, 2516, 2518, 2520, 2522, 2524, 2526, 2528, 2530, 2532,2534, 2526, 2528, 2530, 2532, 2534, 2536, 2538, 2540, 2542, 2544, 2546,2548, 2550, 2552, 2554, 2556, 2558, 2560, 2562, 2564, 2566, 2568, 2570,2572, 2574, 2576, 2578, 2580, 2582, 2584, 2586, 2588, 2590, 2592, 2594,2596, 2598, 2600, 2602, 2604, 2606, 2608, 2610, 2612, 2614, 2616, 2618,2620, 2622, 2624, 2626, 2628, 2630, 2632, 2634, 2636, 2638, 2640, 2642,2644, 2646, 2648, 2650, 2652, 2654, 2656, 2658, 2660, 2662, 2664, 2668,2670, 2672, 2674, 2676, 2678, 2680, 2682, 2684, 2686, 2688, 2690, 2692,2694, 2696, 2698, 2700, 2702, 2704, 2706, 2708, 2710, 2712, 2714, 2716,2718, 2720, 2722, 2724, 2726, 2728, 2730, 2732, 2734, 2736, 2738, 2740,2742, 2744, 2746, 2748, 2750, 2752, 2754, 2756, 2758, 2760, 2762, 2764,2766, 2768, 2770, 2772, 2774, 2776, 2778, 2780, 2782, 2784, 2786, 2788,2790, 2792, 2794, 2796, 2798, 2800, 2802, 2804, 2806, 2808, 2810, 2812,2814, 2816, 2818, 2820, 2822, 2824, 2826, 2828, 2830, 2832, 2834, 2836,2838, 2840, 2842, 2844, 2846, and 2848, wherein the amino acid sequencecomprises one or more amino acid residues selected from the groupconsisting of T at position 2, A or P or S at position 3, V at position4, D at position 6, I or F at position 9, L at position 10, S atposition 13, S at position 14, K at position 15, C at position 16, T orR at position 17, C or S or K at position 20, T at position 24, Q atposition 26, F at position 28, T at position 29, A at position 30, I atposition 31, G at position 33, R at position 34, L at position 35, N atposition 36, H at position 37, Q at position 38, D at position 40, L atposition 44, P at position 45, P or A at position 47, N at position 51,I at position 52, V at position 54, R at position 55, D at position 56,K at position 58, G or D at position 61, V at position 63, L at position70, R at position 72, I at position 75, P at position 76, C at position78, Q at position 80, A or Y at position 82, P at position 83, S or L orV at position 84, A at position 85, W at position 86, R at position 87,E at position 91, D at position 93, Q or G at position 95, N at position96, D at position 99, T at position 100, K at position 107, A atposition 112, T or S or G at position 114, A at position 115, P atposition 117, N at position 120, E at position 121, P at position 122, Rat position 126, V at position 130, S at position 133, A or V atposition 134, L or W or V at position 136, H at position 139, I or R atposition 142, S at position 144, S at position 146, T at position 152, Sor A at position 154, V at position 168, T at position 169, A atposition 174, F at position 177, V at position 178, I at position 180, Gor I at position 181, K at position 184, Y at position 186, S atposition 189, L at position 194, S at position 195, N at position 198, Mat position 199, R at position 203, E at position 215, T at position222, G at position 236, V at position 237, L at position 238, T atposition 240, I or A or V at position 245, Y at position 249, V or I atposition 252, and V at position 254, wherein amino acid residue positionis determined by optimal alignment of the amino acid sequence of theHHDH polypeptide with the reference sequence. Sometimes, these HHDHpolypeptides have an amino acid sequence that is at least 81% identical,82% identical, 83% identical, 84% identical, 85% identical, 86%identical, 87% identical, 88% identical, 89% identical, 90% identical,91% identical 92% identical, 93% identical, 94% identical, 95%,identical, 96% identical, 97% identical, 98% or 99% identical to thereference sequence.

Typically, the reference sequence is selected from the group consistingof SEQ ID NO: 2, SEQ ID NO: 200, SEQ ID NO: 730, SEQ ID NO: 750, SEQ IDNO: 1926, SEQ ID NO: 1984, SEQ ID NO: 2040, SEQ ID NO: 2130, SEQ ID NO:2348, and SEQ ID NO: 2848.

Typically, the amino acid sequences of the HHDH polypeptides describedin paragraphs 031-032 comprise one or more amino acid residues selectedfrom one subgroup selected from (1) T at position 2, A or P or S atposition 3, V at position 4, D at position 6, I or F at position 9, L atposition 10, S at position 13, S at position 14, K at position 15, C atposition 16, T or R at position 17, C or S or K at position 20, T atposition 24, Q at position 26, F at position 28, T at position 29, A atposition 30, I at position 31, G at position 33, R at position 34, L atposition 35, N at position 36, H at F position 37, D at position 40, Lat position 44, P at position 45, P or A at position 47, N at position51, V at position 54, R at position 55, D at position 56, K at position58, G or D at position 61, V at position 63, R at position 72, I atposition 75, P at position 76, C at position 78, Q at position 80, Y atposition 82, S or L at position 84, A at position 85, E at position 91,D at position 93, Q or G at position 95, N at position 96, K at position107, A at position 112, T or S or G at position 114, A at position 115,P at position 117, N at position 120, E at position 121, P at position122, R at position 126, V at position 130, S at position 133, A or V atposition 134, L or W or V at position 136, H at position 139, I or R atposition 142, S at position 144, S at position 146, T at position 152, Sor A at position 154, V at position 168, T at position 169, F atposition 177, V at position 178, I at position 180, G or I at position181, K at position 184, Y at position 186, L at position 194, N atposition 198, M at position 199, E at position 215, G at position 236, Vat position 237, L at position 238, T at position 240, I or A or V atposition 245, Y at position 249, V or I at position 252, and V atposition 254; or (2) Q at position 38, I at position 52, L at position70, A at position 82, P at position 83, V at position 84, W at position86, R at position 87, D at position 99, T at position 100, A at position174, S at position 189, S at position 195, R at position 203, and T atposition 222; or (3) H at position 37, I at position 75, K at position107, A at position 112, A at position 134, A at position 154, V atposition 178, G at position 181, Y at position 186, T at position 222,and V at position 245.

The HHDH polypeptides described in paragraphs 031-033 may have anycombination of two or more, three or more, four or more, five or more,six or more, seven or more, eight or more, nine or more, ten or more,eleven or more, twelve or more, thirteen or more, fourteen or more,fifteen or more, sixteen or more, seventeen more, eighteen or more,nineteen or more, or twenty or more of the above-described amino acidresidues (at the specified positions) in their sequences.

As used herein, the term “isolated” refers to a nucleic acid,polynucleotide, polypeptide, protein, or other component that ispartially or completely separated from components with which it isnormally associated (other proteins, nucleic acids, cells, syntheticreagents, etc.). A nucleic acid (such as a polynucleotide) orpolypeptide is “recombinant” when it is artificial or engineered, orderived from an artificial or engineered protein or nucleic acid. Forexample, a polynucleotide that is inserted into a vector or any otherheterologous location, e.g., in a genome of a recombinant organism, suchthat it is not associated with nucleotide sequences that normally flankthe polynucleotide as it is found in nature is a recombinantpolynucleotide. A protein expressed in vitro or in vivo from arecombinant polynucleotide is an example of a recombinant polypeptide.Likewise, a polynucleotide sequence that does not appear in nature, forexample a variant of a naturally occurring gene, is recombinant.

As used herein, the terms “HHDH activity” and “halohydrin dehalogenaseactivity” are used interchangeably herein to refer to the ability tocatalyze the conversion of ethyl (S)4-chloro-3-hydroxybutyrate (“ECHB”)to a detectable amount of ethyl (R) 4-cyano-3-hydroxybutyrate (“HN”)using the assay described in Example 5A. The term “HHDH polypeptide”refers herein to a polypeptide having HHDH activity. The term “HHDHpolynucleotide” refers to a polynucleotide encoding a polypeptide havingHHDH activity.

The terms “percent identity,” “% identity,” “percent identical,” and “%identical” are used interchangeably herein to refer to the percent aminoacid sequence identity that is obtained by ClustalW analysis (version W1.8 available from European Bioinformatics Institute, Cambridge, UK),counting the number of identical matches in the alignment and dividingsuch number of identical matches by the length of the referencesequence, and using the following default ClustalW parameters to achieveslow/accurate pairwise optimal alignments—Gap Open Penalty: 10; GapExtension Penalty: 0.10; Protein weight matrix: Gonnet series; DNAweight matrix: IUB; Toggle Slow/Fast pairwise alignments=SLOW or FULLAlignment.

Two sequences are “optimally aligned” when they are aligned forsimilarity scoring using a defined amino acid substitution matrix (e.g.,BLOSUM62), gap existence penalty and gap extension penalty so as toarrive at the highest score possible for that pair of sequences. Aminoacid substitution matrices and their use in quantifying the similaritybetween two sequences are well-known in the art. See e.g., Dayhoffet al.(1978), “A model of evolutionary change in proteins”; “Atlas of ProteinSequence and Structure,” Vol. 5, Suppl. 3 (Ed. M. O. Dayhoff), pp.345-352, Natl. Biomed. Res. Round., Washington, D.C.; Henikoffet al.(1992) Proc. Natl. Acad. Sci. USA, 89:10915-10919. The BLOSUM62 matrixis often used as a default scoring substitution matrix in sequencealignment protocols such as Gapped BLAST 2.0. The gap existence penaltyis imposed for the introduction of a single amino acid gap in one of thealigned sequences, and the gap extension penalty is imposed for eachadditional empty amino acid position inserted into an already openedgap. The alignment is defined by the amino acid position of eachsequence at which the alignment begins and ends, and optionally by theinsertion of a gap or multiple gaps in one or both sequences so as toarrive at the highest possible score. While optimal alignment andscoring can be accomplished manually, the process is facilitated by theuse of a computer-implemented alignment algorithm, e.g., gapped BLAST2.0, described in Altschul, et al. (1997) Nucleic Acids Res.,25:3389-3402, and made available to the public at the National Centerfor Biotechnology Information Website. Optimal alignments, includingmultiple alignments can be prepared using readily available programssuch as PSI-BLAST, which is described by Altschul, et al. (1997) NucleicAcids Res., 25:3389-3402.

With respect to an amino acid sequence that is optimally aligned with areference sequence, an amino acid residue “corresponds to” the positionin the reference sequence with which the residue is paired in thealignment. The “position” is denoted by a number that sequentiallyidentifies each amino acid in the reference sequence based on itsposition relative to the N-terminus. Owing to deletions, insertions,truncations, fusions, and the like that must be taken into account whendetermining an optimal alignment, in general the amino acid residuenumber in a test sequence is determined by simply counting from theN-terminal will not necessarily be the same as the number of itscorresponding position in the reference sequence. For example, in a casewhere there is a deletion in an aligned test sequence, there will be noamino acid that corresponds to a position in the reference sequence atthe site of deletion. Where there is an insertion in an alignedreference sequence, that insertion will not correspond to any amino acidposition in the reference sequence. In the case of truncations orfusions there can be stretches of amino acids in either the reference oraligned sequence that do not correspond to any amino acid in thecorresponding sequence.

HHDH polypeptides of the present invention include those encoded by anucleic acid that hybridizes under stringent conditions oversubstantially the entire length of a nucleic acid corresponding to areference nucleic acid sequence selected from the group consisting of:SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67,69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101,103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129,131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157,159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185,187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213,215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241,243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269,271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297,299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325,327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353,355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381,383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409,411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437,439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465,467, 469, 4712, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493,495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521,523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549,551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577,579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605,607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633,635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661,663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689,691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717,719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745,747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771, 773,775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801,803, 805, 807, 809, 811, 813, 815, 817, 819, 821, 823, 825, 827, 829,831, 833, 835, 837, 839, 841, 843, 845, 847, 849, 851, 853, 855, 857,859, 861, 863, 865, 867, 869, 871, 873, 875, 877, 879, 881, 883, 885,887, 889, 891, 893, 895, 897, 899, 901, 903, 905, 907, 909, 911, 913,915, 917, 919, 921, 923, 925, 927, 929, 931, 933, 935, 937, 939, 941,943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969,971, 973, 975, 977, 979, 981, 983, 985, 987, 989, 991, 993, 995, 997,999, 1001, 1103, 1005, 1007, 1009, 1011, 1013, 1015, 1017, 1019, 1021,1023, 1025, 1027, 1029, 1031, 1033, 1035, 1037, 1039, 1041, 1043, 1045,1047, 1049, 1051, 1053, 1055, 1057, 1059, 1061, 1063, 1065, 1067, 1069,1071, 1073, 1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091, 1093,1095, 1097, 1099, 1101, 1103, 1105, 1107, 1109, 1111, 1113, 1115, 1117,1119, 1121, 1123, 1125, 1127, 1129, 1131, 1133, 1135, 1137, 1139, 1141,1143, 1145, 1147, 1149, 1151, 1153, 1155, 1157, 1159, 1161, 1163, 1165,1167, 1169, 1171, 1173, 1175, 1177, 1179, 1181, 1183, 1185, 1187, 1189,1191, 1193, 1195, 1197, 1199, 1201, 1203, 1205, 1207, 1209, 1211, 1213,1215, 1217, 1219, 1221, 1223, 1225, 1227, 1229, 1231, 1233, 1235, 1237,1239, 1241, 1243, 1245, 1247, 1249, 1251, 1253, 1255, 1257, 1259, 1261,1263, 1265, 1267, 1269, 1271, 1273, 1275, 1277, 1279, 1281, 1283, 1285,1287, 1289, 1291, 1293, 1295, 1297, 1299, 1301, 1303, 1305, 1307, 1309,1311, 1313, 1315, 1317, 1319, 1321, 1323, 1325, 1327, 1329, 1331, 1333,1335, 1337, 1339, 1341, 1343, 1345, 1347, 1349, 1351, 1353, 1355, 1357,1359, 1361, 1363, 1365, 1367, 1369, 1371, 1373, 1375, 1377, 1379, 1381,1383, 1385, 1387, 1389, 1391, 1393, 1395, 1397, 1399, 1401, 1403, 1405,1407, 1409, 1411, 1413, 1415, 1417, 1419, 1421, 1423, 1425, 1427, 1429,1431, 1433, 1435, 1437, 1439, 1441, 1443, 1445, 1447, 1449, 1451, 1453,1455, 1457, 1459, 1461, 1463, 1465, 1467, 1469, 1471, 1473, 1475, 1477,1479, 1481, 1483, 1485, 1487, 1489, 1491, 1493, 1495, 1497, 1499, 1501,1503, 1505, 1507, 1509, 1511, 1513, 1515, 1517, 1519, 1521, 1523, 1525,1527, 1529, 1531, 1533, 1535, 1537, 1539, 1541, 1543, 1545, 1547, 1549,1551, 1553, 1555, 1557, 1559, 1561, 1563, 1565, 1567, 1569, 1571, 1573,1575, 1577, 1579, 1581, 1583, 1585, 1587, 1589, 1591, 1593, 1595, 1597,1599, 1601, 1603, 1605, 1607, 1609, 1611, 1613, 1615, 1617, 1619, 1621,1623, 1625, 1627, 1629, 1631, 1633, 1635, 1637, 1639, 1641, 1643, 1645,1647, 1649, 1651, 1653, 1655, 1657, 1659, 1661, 1663, 1665, 1667, 1669,1671, 1673, 1675, 1677, 1679, 1681, 1683, 1685, 1687, 1689, 1691, 1693,1695, 1697, 1699, 1701, 1703, 1705, 1707, 1709, 1711, 1713, 1715, 1717,1719, 1721, 1723, 1725, 1727, 1729, 1731, 1733, 1735, 1737, 1739, 1741,1743, 1745, 1747, 1749, 1751, 1753, 1755, 1757, 1759, 1761, 1763, 1765,1767, 1769, 1771, 1773, 1775, 1777, 1779, 1781, 1783, 1785, 1787, 1789,1791, 1793, 1795, 1797, 1799, 1801, 1803, 1805, 1807, 1809, 1811, 1813,1815, 1817, 1819, 1821, 1823, 1825, 1827, 1829, 1831, 1833, 1835, 1837,1839, 1841, 1843, 1845, 1847, 1849, 1851, 1853, 1855, 1857, 1859, 1861,1863, 1865, 1867, 1869, 1871, 1873, 1875, 1877, 1879, 1881, 1883, 1885,1887, 1889, 1891, 1893, 1895, 1897, 1899, 1901, 1903, 1905, 1907, 1909,1911, 1913, 1915, 1917, 1919, 1921, 1923, 1925, 1927, 1929, 1931, 1933,1935, 1937, 1939, 1941, 1943, 1945, 1947, 1949, 1951, 1953, 1955, 1957,1959, 1961, 1963, 1965, 1967, 1969, 1971, 1973, 1975, 1977, 1979, 1981,1983, 1985, 1987, 1989, 1991, 1993, 1995, 1997, 1999, 2001, 2003, 2005,2007, 2009, 2011, 2013, 2015, 2017, 2019, 2021, 2023, 2025, 2027, 2029,2031, 2033, 2035, 2037, 2039, 2041, 2043, 2045, 2047, 2049, 2051, 2053,2055, 2057, 2059, 2061, 2063, 2065, 2067, 2069, 2071, 2073, 2075, 2077,2079, 2081, 2083, 2085, 2087, 2089, 2091, 2093, 2095, 2097, 2099, 2101,2103, 2105, 2107, 2109, 2111, 2113, 2115, 2117, 2119, 2121, 2123, 2125,2127, 2129, 2131, 2133, 2135, 2137, 2139, 2141, 2143, 2145, 2147, 2149,2151, 2153, 2155, 2157, 2159, 2161, 2163, 2165, 2167, 2169, 2171, 2173,2175, 2177, 2179, 2181, 2183, 2185, 2187, 2189, 2191, 2193, 2195, 2197,2199, 2201, 2203, 2205, 2207, 2209, 2211, 2213, 2215, 2217, 2219, 2221,2223, 2225, 2227, 2229, 2231, 2233, 2235, 2237, 2239, 2241, 2243, 2245,2247, 2249, 2251, 2253, 2255, 2257, 2259, 2261, 2263, 2265, 2267, 2269,2271, 2273, 2275, 2277, 2279, 2281, 2283, 2285, 2287, 2289, 2291, 2293,2295, 2297, 2299, 2301, 2303, 2305, 2307, 2309, 2311, 2313, 2315, 2317,2319, 2321, 2323, 2325, 2327, 2329, 2331, 2333, 2335, 2337, 2339, 2341,2343, 2345, 2347, 2349, 2351, 2353, 2355, 2357, 2359, 2361, 2363, 2365,2367, 2369, 2371, 2373, 2375, 2377, 2379, 2381, 2383, 2385, 2387, 2389,2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2413,2415, 2417, 2419, 2421, 2423, 2425, 2427, 2429, 2431, 2433, 2435, 2437,2439, 2441, 2443, 2445, 2447, 2449, 2451, 2453, 2455, 2457, 2459, 2461,2463, 2465, 2467, 2469, 2471, 2473, 2475, 2477, 2479, 2481, 2483, 2485,2487, 2489, 2491, 2493, 2495, 2497, 2499, 2501, 2503, 2505, 2507, 2509,2511, 2513, 2515, 2517, 2519, 2521, 2523, 2525, 2527, 2529, 2531, 2533,2635, 2537, 2539, 2541, 2543, 2545, 2547, 2549, 2551, 2553, 2555, 2557,2559, 2561, 2563, 2565, 2567, 2569, 2571, 2573, 2575, 2577, 2579, 2581,2583, 2585, 2587, 2589, 2591, 2593, 2595, 2597, 2599, 2601, 2603, 2605,2607, 2609, 2611, 2613, 2615, 2617, 2619, 2621, 2623, 2625, 2627, 2629,2631, 2633, 2635, 2637, 2639, 2641, 2643, 2645, 2647, 2649, 2651, 2653,2655, 2657, 2659, 2661, 2663, 2665, 2667, 2669, 2671, 2673, 2675, 2677,2679, 2681, 2683, 2685, 2687, 2689, 2691, 2693, 2695, 2697, 2699, 2701,2703, 2705, 2707, 2709, 2711, 2713, 2715, 2717, 2719, 2721, 2723, 2725,2727, 2729, 2731, 2733, 2735, 2737, 2739, 2741, 2743, 2745, 2747, 2749,2751, 2753, 2755, 2757, 2759, 2761, 2763, 2765, 2767, 2769, 2771, 2773,2775, 2777, 2779, 2781, 2783, 2785, 2787, 2789, 2791, 2793, 2795, 2797,2799, 2801, 2803, 2805, 2807, 2809, 2811, 2813, 2815, 2817, 2819, 2821,2823, 2825, 2827, 2829, 2831, 2833, 2835, 2837, 2839, 2841, 2843, 2845,2847, and complementary sequences thereof, wherein the encodedpolypeptide has an amino acid sequence comprising one or more amino acidresidues selected from the group consisting of V at position 5, A atposition 27, V at position 45, Q at position 46, I at position 54, V atposition 60, V at position 65, N at position 67, H at position 72, I atposition 77, S at position 87, R at position 91, T at position 93, A atposition 95, E at position 96, M or Q at position 100, T at position103, R or T at position 117, S at position 118, R or I or L or Y atposition 121, S at position 135, R at position 139+E at position 176 orD at position 139+R at position 176, A at position 146, N at position153, H at position 166, G or A at position 177, M or C at position 178,N or D at position 179, D or T at position 180, K at position 181, N atposition 182, V at position 190, A at position 199, W or Y at position201, Y at position 205, L at position 236, T or R at position 238, F atposition 239, V at position 246, E or A or S at position 251, and P atposition 252, wherein amino acid residue position is determined byoptimal alignment with a reference amino acid sequence that is encodedby the reference nucleic acid sequence (i.e., SEQ ID NO: 2, 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82,84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114,116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142,144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170,172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198,200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226,228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254,256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282,284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310,312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338,340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366,368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394,396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422,424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450,452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478,480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506,508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534,536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562,564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590,592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618,620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646,648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674,676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702,704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730,732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758,760, 762, 764, 766, 768, 770, 772, 774, 776, 778, 780, 782, 784, 786,788, 790, 792, 794, 796, 798, 800, 802, 804, 806, 808, 810, 812, 814,816, 818, 820, 822, 824, 826, 828, 830, 832, 834, 836, 838, 840, 842,844, 846, 848, 850, 852, 854, 856, 858, 860, 862, 864, 866, 868, 870,872, 874, 876, 878, 880, 882, 884, 886, 888, 890, 892, 894, 896, 898,900, 902, 904, 906, 908, 910, 912, 914, 916, 918, 920, 922, 924, 926,928, 930, 932, 934, 936, 938, 940, 942, 944, 946, 948, 950, 952, 954,956, 958, 960, 962, 964, 966, 968, 970, 972, 974, 976, 978, 980, 982,984, 986, 988, 990, 992, 994, 996, 998, 1000, 1002, 1004, 1006, 1008,1010, 1012, 1014, 1016, 1018, 1020, 1022, 1024, 1026, 1028, 1030, 1032,1034, 1036, 1038, 1040, 1042, 1044, 1046, 1048, 1050, 1052, 1054, 1056,1058, 1060, 1062, 1064, 1066, 1068, 1070, 1072, 1074, 1076, 1078, 1080,1082, 1084, 1086, 1088, 1090, 1092, 1094, 1096, 1098, 1100, 1102, 1104,1106, 1108, 1110, 1112, 1114, 1116, 1118, 1120, 1122, 1124, 1126, 1128,1130, 1132, 1134, 1136, 1138, 1140, 1142, 1144, 1146, 1148, 1150, 1152,1154, 1156, 1158, 1160, 1162, 1164, 1166, 1168, 1170, 1172, 1174, 1176,1178, 1180, 1182, 1184, 1186, 1188, 1190, 1192, 1194, 1196, 1198, 1200,1202, 1204, 1206, 1208, 1210, 1212, 1214, 1216, 1218, 1220, 1222, 1224,1226, 1228, 1230, 1232, 1234, 1236, 1238, 1240, 1242, 1244, 1246, 1248,1250, 1252, 1254, 1256, 1258, 1260, 1262, 1264, 1266, 1268, 1270, 1272,1274, 1276, 1278, 1280, 1282, 1284, 1286, 1288, 1290, 1292, 1294, 1296,1298, 1300, 1302, 1304, 1306, 1308, 1310, 1312, 1314, 1316, 1318, 1320,1322, 1324, 1326, 1328, 1330, 1332, 1334, 1336, 1338, 1340, 1342, 1344,1346, 1348, 1350, 1352, 1354, 1356, 1358, 1360, 1362, 1364, 1366, 1368,1370, 1372, 1374, 1376, 1378, 1380, 1382, 1384, 1386, 1388, 1390, 1392,1394, 1396, 1398, 1400, 1402, 1404, 1406, 1408, 1410, 1412, 1414, 1416,1418, 1420, 1422, 1424, 1426, 1428, 1430, 1432, 1434, 1436, 1438, 1440,1442, 1444, 1446, 1448, 1450, 1452, 1454, 1456, 1458, 1460, 1462, 1464,1466, 1468, 1470, 1472, 1474, 1476, 1478, 1480, 1482, 1484, 1486, 1488,1490, 1492, 1494, 1496, 1498, 1500, 1502, 1504, 1506, 1508, 1510, 1512,1514, 1516, 1518, 1520, 1522, 1524, 1526, 1528, 1530, 1532, 1534, 1536,1538, 1540, 1542, 1544, 1546, 1548, 1550, 1552, 1554, 1556, 1558, 1560,1562, 1564, 1566, 1568, 1570, 1572, 1574, 1576, 1578, 1580, 1582, 1584,1586, 1588, 1590, 1592, 1594, 1596, 1598, 1600, 1602, 1604, 1606, 1608,1610, 1612, 1614, 1616, 1618, 1620, 1622, 1624, 1626, 1628, 1630, 1632,1634, 1636, 1638, 1640, 1642, 1644, 1646, 1648, 1650, 1652, 1654, 1656,1658, 1660, 1662, 1664, 1666, 1668, 1670, 1672, 1674, 1676, 1678, 1680,1682, 1684, 1686, 1688, 1690, 1692, 1694, 1696, 1698, 1700, 1702, 1704,1706, 1708, 1710, 1712, 1714, 1716, 1718, 1720, 1722, 1724, 1726, 1728,1730, 1732, 1734, 1736, 1738, 1740, 1742, 1744, 1746, 1748, 1750, 1752,1754, 1756, 1758, 1760, 1762, 1764, 1766, 1768, 1770, 1772, 1774, 1776,1778, 1780, 1782, 1784, 1786, 1788, 1790, 1792, 1794, 1796, 1798, 1800,1802, 1804, 1806, 1808, 1810, 1812, 1814, 1816, 1818, 1820, 1822, 1824,1826, 1828, 1830, 1832, 1834, 1836, 1838, 1840, 1842, 1844, 1846, 1848,1850, 1852, 1854, 1856, 1858, 1860, 1862, 1864, 1866, 1868, 1870, 1872,1874, 1876, 1878, 1880, 1882, 1884, 1886, 1888, 1890, 1892, 1894, 1896,1898, 1900, 1902, 1904, 1906, 1908, 1910, 1912, 1914, 1916, 1918, 1920,1922, 1924, 1926, 1928, 1930, 1932, 1934, 1936, 1938, 1940, 1942, 1944,1946, 1948, 1950, 1952, 1954, 1956, 1958, 1960, 1962, 1964, 1966, 1968,1970, 1972, 1974, 1976, 1978, 1980, 1982, 1984, 1986, 1988, 1990, 1992,1994, 1996, 1998, 2000, 2002, 2004, 2006, 2008, 2010, 2012, 2014, 2016,2018, 2020, 2022, 2024, 2026, 2028, 2030, 2032, 2034, 2036, 2038, 2040,2042, 2044, 2046, 2048, 2050, 2052, 2054, 2056, 2058, 2060, 2062, 2064,2066, 2068, 2070, 2072, 2074, 2076, 2078, 2080, 2082, 2084, 2086, 2088,2090, 2092, 2094, 2096, 2098, 2100, 2102, 2104, 2106, 2108, 2110, 2112,2114, 2116, 2118, 2120, 2122, 2124, 2126, 2128, 2130, 2132, 2134, 2136,2138, 2140, 2142, 2144, 2146, 2148, 2150, 2152, 2154, 2156, 2158, 2160,2162, 2164, 2166, 2168, 2170, 2172, 2174, 2176, 2178, 2180, 2182, 2184,2186, 2188, 2190, 2192, 2194, 2196, 2198, 2200, 2202, 2204, 2206, 2208,2210, 2212, 2214, 2216, 2218, 2220, 2222, 2224, 2226, 2228, 2230, 2232,2234, 2236, 2238, 2240, 2242, 2244, 2246, 2248, 2250, 2252, 2254, 2256,2258, 2260, 2262, 2264, 2266, 2268, 2270, 2272, 2274, 2276, 2278, 2280,2282, 2284, 2286, 2288, 2290, 2292, 2294, 2296, 2298, 2300, 2302, 2304,2306, 2308, 2310, 2312, 2314, 2316, 2318, 2320, 2322, 2324, 2326, 2328,2330, 2332, 2334, 2336, 2338, 2340, 2342, 2344, 2346, 2348, 2350, 2352,2354, 2356, 2358, 2360, 2362, 2364, 2366, 2368, 2370, 2372, 2374, 2376,2378, 2380, 2382, 2384, 2386, 2388, 2390, 2392, 2394, 2396, 2398, 2400,2402, 2404, 2406, 2408, 2410, 2412, 2414, 2416, 2418, 2420, 2422, 2424,2426, 2428, 2430, 2432, 2434, 2436, 2438, 2440, 2442, 2444, 2446, 2448,2450, 2452, 2454, 2456, 2458, 2460, 2462, 2464, 2466, 2468, 2470, 2472,2474, 2476, 2478, 2480, 2482, 2484, 2486, 2488, 2490, 2492, 2494, 2496,2498, 2500, 2502, 2504, 2506, 2508, 2510, 2512, 2514, 2516, 2518, 2520,2522, 2524, 2526, 2528, 2530, 2532, 2534, 2536, 2538, 2540, 2542, 2544,2546, 2548, 2550, 2552, 2554, 2556, 2558, 2560, 2562, 2564, 2566, 2568,2570, 2572, 2574, 2576, 2578, 2580, 2582, 2584, 2586, 2588, 2590, 2592,2594, 2596, 2598, 2600, 2602, 2604, 2606, 2608, 2610, 2612, 2614, 2616,2618, 2620, 2622, 2624, 2626, 2628, 2630, 2632, 2634, 2636, 2638, 2640,2642, 2644, 2646, 2648, 2650, 2652, 2654, 2656, 2658, 2660, 2662, 2664,2666, 2668, 2670, 2672, 2674, 2676, 2678, 2680, 2682, 2684, 2686, 2688,2690, 2692, 2694, 2696, 2698, 2700, 2702, 2704, 2706, 2708, 2710, 2712,2714, 2716, 2718, 2720, 2722, 2724, 2726, 2728, 2730, 2732, 2734, 2736,2738, 2740, 2742, 2744, 2746, 2748, 2750, 2752, 2754, 2756, 2758, 2760,2762, 2764, 2766, 2768, 2770, 2772, 2774, 2776, 2778, 2780, 2782, 2784,2786, 2788, 2790, 2792, 2794, 2796, 2798, 2800, 2802, 2804, 2806, 2808,2810, 2812, 2814, 2816, 2818, 2820, 2822, 2824, 2826, 2828, 2830, 2832,2834, 2836, 2838, 2840, 2842, 2844, 2846, and 2848, respectively).

In some embodiments, the above-described HHDH polypeptides are encodedby an amino acid sequence that comprises one or more amino acid residuesselected from the subgroup consisting of A at position 27, Q at position46, I at position 54, V at position 60, V at position 65, S at position87, R at position 91, A at position 95, E at position 96, M or Q atposition 100, R at position 117, S at position 118, R at position 121, Sat position 135, A at position 146, N at position 153, M at position178, N at position 179, N at position 182, V at position 190, A atposition 199, Y at position 201, Y at position 205, L at position 236, Tat position 238, and E or A at position 251.

In certain embodiments, these HHDH polypeptides are encoded by an aminoacid sequence having one or more amino acid residues selected from thesubgroup consisting of V at position 5, V at position 45, N at position67, H at position 72, I at position 77, T at position 93, T at position103, T at position 117, S at position 118, I or L or Y at position 121,R at position 139+E at position 176 or D at position 139+R at position176, H at position 166, G or A at position 177, C at position 178, D atposition 179, W at position 201, R at position 238, F at position 239, Vat position 246, S at position 251, and P at position 252. In otherembodiments, these HHDH polypeptides are encoded by an amino acidsequence that comprises one or more residues selected from the groupconsisting of I at position 54, V at position 60, N at position 67, H atposition 72, T at position 93, M at position 100, T at position 117, Rat position 121, S at position 135, A at position 146, A or G atposition 177, M or C at position 178, D at position 179, W at position201, Y at position 205, R at position 238, F at position 239, and V atposition 246. Some of these HHDH polypeptides have an amino acidsequence that comprises one or more amino acid residues selected fromthe subgroup group consisting of N at position 67, H at position 72, Tat position 117, R at position 121, S at position 135, A at position146, A at position 177, D at position 179, W at position 201, Y atposition 205, and V at position 246. HHDH polypeptides of the presentinvention include those having an amino acid sequence comprising thefollowing combination of amino acid residues: N at position 67+H atposition 72+T at position 117+R at position 121+S at position 135+A atposition 146+A at position 154+A at position 177+D at position 179+W atposition 201+Y at position 205+V at position 246. In other embodimentsthe HHDH polypeptides have an amino acid sequence that comprises one ormore amino acid residues selected from the group consisting of I atposition 54, V at position 60, E at position 91, T at position 93, M atposition 100, G at position 177, M or C at position 178, R at position238, and F at position 239.

In addition to the amino acid residues described above, the HHDHpolypeptides described in paragraphs 040-042 may further comprise one ormore amino acid residues selected from the group consisting of T atposition 2, A or P or S at position 3, V at position 4, D at position 6,I or F at position 9, L at position 10, S at position 13, S at position14, K at position 15, C at position 16, T or R at position 17, C or S orK at position 20, T at position 24, Q at position 26, F at position 28,T at position 29, A at position 30, I at position 31, G at position 33,R at position 34, L at position 35, N at position 36, H at position 37,Q at position 38, D at position 40, L at position 44, P at position 45(if not V from paragraphs 040-042), P or A at position 47, N at position51, I at position 52, V at position 54 (if not I), R at position 55, Dat position 56, K at position 58, G or D at position 61, V at position63, L at position 70, R at position 72 (if not H), I at position 75, Pat position 76, C at position 78, Q at position 80, A or Y at position82, P at position 83, S or L or V at position 84, A at position 85, W atposition 86, R at position 87, E at position 91 (if not R), D atposition 93 (if not T), Q or G at position 95 (if not A), N at position96 (if not E), D at position 99, T at position 100, K at position 107, Aat position 112, T or S or G at position 114, A at position 115, P atposition 117 (if not R or T), N at position 120, E at position 121 (ifnot R or I or L or Y), P at position 122, R at position 126, V atposition 130, S at position 133, A or V at position 134, L or W or V atposition 136, H at position 139 (if not R or D), I or R at position 142,S at position 144, S at position 146 (if not A), T at position 152, S orA at position 154, V at position 168, T at position 169, A at position174, F at position 177 (if not G or A), V at position 178 (if not M orC), I at position 180, G or I at position 181, K at position 184, Y atposition 186, S at position 189, L at position 194, S at position 195, Nat position 198, M at position 199 (if not A), R at position 203, E atposition 215, T at position 222, G at position 236 (if not L), V atposition 237, L at position 238 (if not T or R), T at position 240, I orA or V at position 245, Y at position 249, V or I at position 252 (ifnot P), and V at position 254.

Some of the HHDH polypeptides described in paragraphs 040-042 furthercomprise one or more amino acid residues selected from one of thefollowing subgroups: (1) T at position 2, A or P or S at position 3, Vat position 4, D at position 6, I or F at position 9, L at position 10,S at position 13, S at position 14, K at position 15, C at position 16,T or R at position 17, C or S or K at position 20, T at position 24, Qat position 26, F at position 28, T at position 29, A at position 30, Iat position 31, G at position 33, R at position 34, L at position 35, Nat position 36, H at position 37, D at position 40, L at position 44, Pat position 45 (if not I), P or A at position 47, N at position 51, V atposition 54 (if not I), R at position 55, D at position 56, K atposition 58, G or D at position 61, V at position 63, R at position 72(if not H), I at position 75, P at position 76, C at position 78, Q atposition 80, Y at position 82, S or L at position 84, A at position 85,E at position 91 (if not R), D at position 93 (if not T), Q or G atposition 95 (if not A), N at position 96 (if not E), K at position 107,A at position 112, T or S or G at position 114, A at position 115, P atposition 117 (if not R or T), N at position 120, E at position 121 (ifnot R or I or L or Y), P at position 122, R at position 126, V atposition 130, S at position 133, A or V at position 134, L or W or V atposition 136, H at position 139 (if not R or D), I or R at position 142,S at position 144, S at position 146 (if not A), T at position 152, S orA at position 154, V at position 168, T at position 169, F at position177 (if not G or A), V at position 178 (if not M or C), I at position180, G or I at position 181, K at position 184, Y at position 186, L atposition 194, N at position 198, M at position 199 (if not A), E atposition 215, G at position 236, V at position 237, L at position 238(if not T or R), T at position 240, I or A or V at position 245, Y atposition 249, V or I at position 252 (if not P), and V at position 254;or (2) Q at position 38, I at position 52, L at position 70, A atposition 82, P at position 83, V at position 84, W at position 86, R atposition 87, D at position 99, T at position 100, A at position 174, Sat position 189, S at position 195, R at position 203, and T at position222; or (2) H at position 37, I at position 75, K at position 107, A atposition 112, A at position 134, A at position 154, V at position 178(if not M or C), G at position 181, Y at position 186, T at position222, and V at position 245. The parentheticals in the above paragraphs(“if not X”) refers to the proviso in which the specified residue isexcluded from the specified amino acid residue position if amino acidresidue X is selected from paragraphs 040-042 for that specified residueposition.

HHDH polypeptides of the present invention include those encoded by anucleic acid that hybridizes under stringent conditions oversubstantially the entire length of a nucleic acid corresponding to areference nucleic acid sequence selected from the group consisting of:SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67,69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101,103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129,131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157,159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185,187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213,215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241,243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269,271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297,299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325,327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353,355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381,383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409,411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437,439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465,467, 469, 4712, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493,495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521,523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549,551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577,579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605,607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633,635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661,663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689,691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717,719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745,747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771, 773,775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801,803, 805, 807, 809, 811, 813, 815, 817, 819, 821, 823, 825, 827, 829,831, 833, 835, 837, 839, 841, 843, 845, 847, 849, 851, 853, 855, 857,859, 861, 863, 865, 867, 869, 871, 873, 875, 877, 879, 881, 883, 885,887, 889, 891, 893, 895, 897, 899, 901, 903, 905, 907, 909, 911, 913,915, 917, 919, 921, 923, 925, 927, 929, 931, 933, 935, 937, 939, 941,943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969,971, 973, 975, 977, 979, 981, 983, 985, 987, 989, 991, 993, 995, 997,999, 1001, 1103, 1005, 1007, 1009, 1011, 1013, 1015, 1017, 1019, 1021,1023, 1025, 1027, 1029, 1031, 1033, 1035, 1037, 1039, 1041, 1043, 1045,1047, 1049, 1051, 1053, 1055, 1057, 1059, 1061, 1063, 1065, 1067, 1069,1071, 1073, 1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091, 1093,1095, 1097, 1099, 1101, 1103, 1105, 1107, 1109, 1111, 1113, 1115, 1117,1119, 1121, 1123, 1125, 1127, 1129, 1131, 1133, 1135, 1137, 1139, 1141,1143, 1145, 1147, 1149, 1151, 1153, 1155, 1157, 1159, 1161, 1163, 1165,1167, 1169, 1171, 1173, 1175, 1177, 1179, 1181, 1183, 1185, 1187, 1189,1191, 1193, 1195, 1197, 1199, 1201, 1203, 1205, 1207, 1209, 1211, 1213,1215, 1217, 1219, 1221, 1223, 1225, 1227, 1229, 1231, 1233, 1235, 1237,1239, 1241, 1243, 1245, 1247, 1249, 1251, 1253, 1255, 1257, 1259, 1261,1263, 1265, 1267, 1269, 1271, 1273, 1275, 1277, 1279, 1281, 1283, 1285,1287, 1289, 1291, 1293, 1295, 1297, 1299, 1301, 1303, 1305, 1307, 1309,1311, 1313, 1315, 1317, 1319, 1321, 1323, 1325, 1327, 1329, 1331, 1333,1335, 1337, 1339, 1341, 1343, 1345, 1347, 1349, 1351, 1353, 1355, 1357,1359, 1361, 1363, 1365, 1367, 1369, 1371, 1373, 1375, 1377, 1379, 1381,1383, 1385, 1387, 1389, 1391, 1393, 1395, 1397, 1399, 1401, 1403, 1405,1407, 1409, 1411, 1413, 1415, 1417, 1419, 1421, 1423, 1425, 1427, 1429,1431, 1433, 1435, 1437, 1439, 1441, 1443, 1445, 1447, 1449, 1451, 1453,1455, 1457, 1459, 1461, 1463, 1465, 1467, 1469, 1471, 1473, 1475, 1477,1479, 1481, 1483, 1485, 1487, 1489, 1491, 1493, 1495, 1497, 1499, 1501,1503, 1505, 1507, 1509, 1511, 1513, 1515, 1517, 1519, 1521, 1523, 1525,1527, 1529, 1531, 1533, 1535, 1537, 1539, 1541, 1543, 1545, 1547, 1549,1551, 1553, 1555, 1557, 1559, 1561, 1563, 1565, 1567, 1569, 1571, 1573,1575, 1577, 1579, 1581, 1583, 1585, 1587, 1589, 1591, 1593, 1595, 1597,1599, 1601, 1603, 1605, 1607, 1609, 1611, 1613, 1615, 1617, 1619, 1621,1623, 1625, 1627, 1629, 1631, 1633, 1635, 1637, 1639, 1641, 1643, 1645,1647, 1649, 1651, 1653, 1655, 1657, 1659, 1661, 1663, 1665, 1667, 1669,1671, 1673, 1675, 1677, 1679, 1681, 1683, 1685, 1687, 1689, 1691, 1693,1695, 1697, 1699, 1701, 1703, 1705, 1707, 1709, 1711, 1713, 1715, 1717,1719, 1721, 1723, 1725, 1727, 1729, 1731, 1733, 1735, 1737, 1739, 1741,1743, 1745, 1747, 1749, 1751, 1753, 1755, 1757, 1759, 1761, 1763, 1765,1767, 1769, 1771, 1773, 1775, 1777, 1779, 1781, 1783, 1785, 1787, 1789,1791, 1793, 1795, 1797, 1799, 1801, 1803, 1805, 1807, 1809, 1811, 1813,1815, 1817, 1819, 1821, 1823, 1825, 1827, 1829, 1831, 1833, 1835, 1837,1839, 1841, 1843, 1845, 1847, 1849, 1851, 1853, 1855, 1857, 1859, 1861,1863, 1865, 1867, 1869, 1871, 1873, 1875, 1877, 1879, 1881, 1883, 1885,1887, 1889, 1891, 1893, 1895, 1897, 1899, 1901, 1903, 1905, 1907, 1909,1911, 1913, 1915, 1917, 1919, 1921, 1923, 1925, 1927, 1929, 1931, 1933,1935, 1937, 1939, 1941, 1943, 1945, 1947, 1949, 1951, 1953, 1955, 1957,1959, 1961, 1963, 1965, 1967, 1969, 1971, 1973, 1975, 1977, 1979, 1981,1983, 1985, 1987, 1989, 1991, 1993, 1995, 1997, 1999, 2001, 2003, 2005,2007, 2009, 2011, 2013, 2015, 2017, 2019, 2021, 2023, 2025, 2027, 2029,2031, 2033, 2035, 2037, 2039, 2041, 2043, 2045, 2047, 2049, 2051, 2053,2055, 2057, 2059, 2061, 2063, 2065, 2067, 2069, 2071, 2073, 2075, 2077,2079, 2081, 2083, 2085, 2087, 2089, 2091, 2093, 2095, 2097, 2099, 2101,2103, 2105, 2107, 2109, 2111, 2113, 2115, 2117, 2119, 2121, 2123, 2125,2127, 2129, 2131, 2133, 2135, 2137, 2139, 2141, 2143, 2145, 2147, 2149,2151, 2153, 2155, 2157, 2159, 2161, 2163, 2165, 2167, 2169, 2171, 2173,2175, 2177, 2179, 2181, 2183, 2185, 2187, 2189, 2191, 2193, 2195, 2197,2199, 2201, 2203, 2205, 2207, 2209, 2211, 2213, 2215, 2217, 2219, 2221,2223, 2225, 2227, 2229, 2231, 2233, 2235, 2237, 2239, 2241, 2243, 2245,2247, 2249, 2251, 2253, 2255, 2257, 2259, 2261, 2263, 2265, 2267, 2269,2271, 2273, 2275, 2277, 2279, 2281, 2283, 2285, 2287, 2289, 2291, 2293,2295, 2297, 2299, 2301, 2303, 2305, 2307, 2309, 2311, 2313, 2315, 2317,2319, 2321, 2323, 2325, 2327, 2329, 2331, 2333, 2335, 2337, 2339, 2341,2343, 2345, 2347, 2349, 2351, 2353, 2355, 2357, 2359, 2361, 2363, 2365,2367, 2369, 2371, 2373, 2375, 2377, 2379, 2381, 2383, 2385, 2387, 2389,2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2413,2415, 2417, 2419, 2421, 2423, 2425, 2427, 2429, 2431, 2433, 2435, 2437,2439, 2441, 2443, 2445, 2447, 2449, 2451, 2453, 2455, 2457, 2459, 2461,2463, 2465, 2467, 2469, 2471, 2473, 2475, 2477, 2479, 2481, 2483, 2485,2487, 2489, 2491, 2493, 2495, 2497, 2499, 2501, 2503, 2505, 2507, 2509,2511, 2513, 2515, 2517, 2519, 2521, 2523, 2525, 2527, 2529, 2531, 2533,2635, 2537, 2539, 2541, 2543, 2545, 2547, 2549, 2551, 2553, 2555, 2557,2559, 2561, 2563, 2565, 2567, 2569, 2571, 2573, 2575, 2577, 2579, 2581,2583, 2585, 2587, 2589, 2591, 2593, 2595, 2597, 2599, 2601, 2603, 2605,2607, 2609, 2611, 2613, 2615, 2617, 2619, 2621, 2623, 2625, 2627, 2629,2631, 2633, 2635, 2637, 2639, 2641, 2643, 2645, 2647, 2649, 2651, 2653,2655, 2657, 2659, 2661, 2663, 2665, 2667, 2669, 2671, 2673, 2675, 2677,2679, 2681, 2683, 2685, 2687, 2689, 2691, 2693, 2695, 2697, 2699, 2701,2703, 2705, 2707, 2709, 2711, 2713, 2715, 2717, 2719, 2721, 2723, 2725,2727, 2729, 2731, 2733, 2735, 2737, 2739, 2741, 2743, 2745, 2747, 2749,2751, 2753, 2755, 2757, 2759, 2761, 2763, 2765, 2767, 2769, 2771, 2773,2775, 2777, 2779, 2781, 2783, 2785, 2787, 2789, 2791, 2793, 2795, 2797,2799, 2801, 2803, 2805, 2807, 2809, 2811, 2813, 2815, 2817, 2819, 2821,2823, 2825, 2827, 2829, 2831, 2833, 2835, 2837, 2839, 2841, 2843, 2845,2847, and complementary sequences thereof, wherein the encodedpolypeptide has an amino acid sequence comprising one or more amino acidresidues selected from the group consisting of T at position 2, A or Por S at position 3, V at position 4, D at position 6, I or F at position9, L at position 10, S at position 13, S at position 14, K at position15, C at position 16, T or R at position 17, C or S or K at position 20,T at position 24, Q at position 26, F at position 28, T at position 29,A at position 30, I at position 31, G at position 33, R at position 34,L at position 35, N at position 36, H at position 37, Q at position 38,D at position 40, L at position 44, P at position 45, P or A at position47, N at position 51, I at position 52, V at position 54, R at position55, D at position 56, K at position 58, G or D at position 61, V atposition 63, L at position 70, R at position 72, I at position 75, P atposition 76, C at position 78, Q at position 80, A or Y at position 82,P at position 83, S or L or V at position 84, A at position 85, W atposition 86, R at position 87, E at position 91, D at position 93, Q orG at position 95, N at position 96, D at position 99, T at position 100,K at position 107, A at position 112, T or S or G at position 114, A atposition 115, P at position 117, N at position 120, E at position 121, Pat position 122, R at position 126, V at position 130, S at position133, A or V at position 134, L or W or V at position 136, H at position139, I or R at position 142, S at position 144, S at position 146, T atposition 152, S or A at position 154, V at position 168, T at position169, A at position 174, F at position 177, V at position 178, I atposition 180, G or I at position 181, K at position 184, Y at position186, S at position 189, L at position 194, S at position 195, N atposition 198, M at position 199, R at position 203, E at position 215, Tat position 222, G at position 236, V at position 237, L at position238, T at position 240, I or A or V at position 245, Y at position 249,V or I at position 252, and V at position 254, and wherein amino acidresidue position is determined by optimal alignment with a referenceamino acid sequence that is encoded by the reference nucleic acidsequence (i.e., SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96,98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152,154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180,182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208,210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236,238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264,266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292,294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320,322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348,350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376,378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404,406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432,434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460,462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488,490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516,518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544,546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572,574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600,602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628,630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656,658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684,686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712,714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740,742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768,770, 772, 774, 776, 778, 780, 782, 784, 786, 788, 790, 792, 794, 796,798, 800, 802, 804, 806, 808, 810, 812, 814, 816, 818, 820, 822, 824,826, 828, 830, 832, 834, 836, 838, 840, 842, 844, 846, 848, 850, 852,854, 856, 858, 860, 862, 864, 866, 868, 870, 872, 874, 876, 878, 880,882, 884, 886, 888, 890, 892, 894, 896, 898, 900, 902, 904, 906, 908,910, 912, 914, 916, 918, 920, 922, 924, 926, 928, 930, 932, 934, 936,938, 940, 942, 944, 946, 948, 950, 952, 954, 956, 958, 960, 962, 964,966, 968, 970, 972, 974, 976, 978, 980, 982, 984, 986, 988, 990, 992,994, 996, 998, 1000, 1002, 1004, 1006, 1008, 1010, 1012, 1014, 1016,1018, 1020, 1022, 1024, 1026, 1028, 1030, 1032, 1034, 1036, 1038, 1040,1042, 1044, 1046, 1048, 1050, 1052, 1054, 1056, 1058, 1060, 1062, 1064,1066, 1068, 1070, 1072, 1074, 1076, 1078, 1080, 1082, 1084, 1086, 1088,1090, 1092, 1094, 1096, 1098, 1100, 1102, 1104, 1106, 1108, 1110, 1112,1114, 1116, 1118, 1120, 1122, 1124, 1126, 1128, 1130, 1132, 1134, 1136,1138, 1140, 1142, 1144, 1146, 1148, 1150, 1152, 1154, 1156, 1158, 1160,1162, 1164, 1166, 1168, 1170, 1172, 1174, 1176, 1178, 1180, 1182, 1184,1186, 1188, 1190, 1192, 1194, 1196, 1198, 1200, 1202, 1204, 1206, 1208,1210, 1212, 1214, 1216, 1218, 1220, 1222, 1224, 1226, 1228, 1230, 1232,1234, 1236, 1238, 1240, 1242, 1244, 1246, 1248, 1250, 1252, 1254, 1256,1258, 1260, 1262, 1264, 1266, 1268, 1270, 1272, 1274, 1276, 1278, 1280,1282, 1284, 1286, 1288, 1290, 1292, 1294, 1296, 1298, 1300, 1302, 1304,1306, 1308, 1310, 1312, 1314, 1316, 1318, 1320, 1322, 1324, 1326, 1328,1330, 1332, 1334, 1336, 1338, 1340, 1342, 1344, 1346, 1348, 1350, 1352,1354, 1356, 1358, 1360, 1362, 1364, 1366, 1368, 1370, 1372, 1374, 1376,1378, 1380, 1382, 1384, 1386, 1388, 1390, 1392, 1394, 1396, 1398, 1400,1402, 1404, 1406, 1408, 1410, 1412, 1414, 1416, 1418, 1420, 1422, 1424,1426, 1428, 1430, 1432, 1434, 1436, 1438, 1440, 1442, 1444, 1446, 1448,1450, 1452, 1454, 1456, 1458, 1460, 1462, 1464, 1466, 1468, 1470, 1472,1474, 1476, 1478, 1480, 1482, 1484, 1486, 1488, 1490, 1492, 1494, 1496,1498, 1500, 1502, 1504, 1506, 1508, 1510, 1512, 1514, 1516, 1518, 1520,1522, 1524, 1526, 1528, 1530, 1532, 1534, 1536, 1538, 1540, 1542, 1544,1546, 1548, 1550, 1552, 1554, 1556, 1558, 1560, 1562, 1564, 1566, 1568,1570, 1572, 1574, 1576, 1578, 1580, 1582, 1584, 1586, 1588, 1590, 1592,1594, 1596, 1598, 1600, 1602, 1604, 1606, 1608, 1610, 1612, 1614, 1616,1618, 1620, 1622, 1624, 1626, 1628, 1630, 1632, 1634, 1636, 1638, 1640,1642, 1644, 1646, 1648, 1650, 1652, 1654, 1656, 1658, 1660, 1662, 1664,1666, 1668, 1670, 1672, 1674, 1676, 1678, 1680, 1682, 1684, 1686, 1688,1690, 1692, 1694, 1696, 1698, 1700, 1702, 1704, 1706, 1708, 1710, 1712,1714, 1716, 1718, 1720, 1722, 1724, 1726, 1728, 1730, 1732, 1734, 1736,1738, 1740, 1742, 1744, 1746, 1748, 1750, 1752, 1754, 1756, 1758, 1760,1762, 1764, 1766, 1768, 1770, 1772, 1774, 1776, 1778, 1780, 1782, 1784,1786, 1788, 1790, 1792, 1794, 1796, 1798, 1800, 1802, 1804, 1806, 1808,1810, 1812, 1814, 1816, 1818, 1820, 1822, 1824, 1826, 1828, 1830, 1832,1834, 1836, 1838, 1840, 1842, 1844, 1846, 1848, 1850, 1852, 1854, 1856,1858, 1860, 1862, 1864, 1866, 1868, 1870, 1872, 1874, 1876, 1878, 1880,1882, 1884, 1886, 1888, 1890, 1892, 1894, 1896, 1898, 1900, 1902, 1904,1906, 1908, 1910, 1912, 1914, 1916, 1918, 1920, 1922, 1924, 1926, 1928,1930, 1932, 1934, 1936, 1938, 1940, 1942, 1944, 1946, 1948, 1950, 1952,1954, 1956, 1958, 1960, 1962, 1964, 1966, 1968, 1970, 1972, 1974, 1976,1978, 1980, 1982, 1984, 1986, 1988, 1990, 1992, 1994, 1996, 1998, 2000,2002, 2004, 2006, 2008, 2010, 2012, 2014, 2016, 2018, 2020, 2022, 2024,2026, 2028, 2030, 2032, 2034, 2036, 2038, 2040, 2042, 2044, 2046, 2048,2050, 2052, 2054, 2056, 2058, 2060, 2062, 2064, 2066, 2068, 2070, 2072,2074, 2076, 2078, 2080, 2082, 2084, 2086, 2088, 2090, 2092, 2094, 2096,2098, 2100, 2102, 2104, 2106, 2108, 2110, 2112, 2114, 2116, 2118, 2120,2122, 2124, 2126, 2128, 2130, 2132, 2134, 2136, 2138, 2140, 2142, 2144,2146, 2148, 2150, 2152, 2154, 2156, 2158, 2160, 2162, 2164, 2166, 2168,2170, 2172, 2174, 2176, 2178, 2180, 2182, 2184, 2186, 2188, 2190, 2192,2194, 2196, 2198, 2200, 2202, 2204, 2206, 2208, 2210, 2212, 2214, 2216,2218, 2220, 2222, 2224, 2226, 2228, 2230, 2232, 2234, 2236, 2238, 2240,2242, 2244, 2246, 2248, 2250, 2252, 2254, 2256, 2258, 2260, 2262, 2264,2266, 2268, 2270, 2272, 2274, 2276, 2278, 2280, 2282, 2284, 2286, 2288,2290, 2292, 2294, 2296, 2298, 2300, 2302, 2304, 2306, 2308, 2310, 2312,2314, 2316, 2318, 2320, 2322, 2324, 2326, 2328, 2330, 2332, 2334, 2336,2338, 2340, 2342, 2344, 2346, 2348, 2350, 2352, 2354, 2356, 2358, 2360,2362, 2364, 2366, 2368, 2370, 2372, 2374, 2376, 2378, 2380, 2382, 2384,2386, 2388, 2390, 2392, 2394, 2396, 2398, 2400, 2402, 2404, 2406, 2408,2410, 2412, 2414, 2416, 2418, 2420, 2422, 2424, 2426, 2428, 2430, 2432,2434, 2436, 2438, 2440, 2442, 2444, 2446, 2448, 2450, 2452, 2454, 2456,2458, 2460, 2462, 2464, 2466, 2468, 2470, 2472, 2474, 2476, 2478, 2480,2482, 2484, 2486, 2488, 2490, 2492, 2494, 2496, 2498, 2500, 2502, 2504,2506, 2508, 2510, 2512, 2514, 2516, 2518, 2520, 2522, 2524, 2526, 2528,2530, 2532, 2534, 2536, 2538, 2540, 2542, 2544, 2546, 2548, 2550, 2552,2554, 2556, 2558, 2560, 2562, 2564, 2566, 2568, 2570, 2572, 2574, 2576,2578, 2580, 2582, 2584, 2586, 2588, 2590, 2592, 2594, 2596, 2598, 2600,2602, 2604, 2606, 2608, 2610, 2612, 2614, 2616, 2618, 2620, 2622, 2624,2626, 2628, 2630, 2632, 2634, 2636, 2638, 2640, 2642, 2644, 2646, 2648,2650, 2652, 2654, 2656, 2658, 2660, 2662, 2664, 2666, 2668, 2670, 2672,2674, 2676, 2678, 2680, 2682, 2684, 2686, 2688, 2690, 2692, 2694, 2696,2698, 2700, 2702, 2704, 2706, 2708, 2710, 2712, 2714, 2716, 2718, 2720,2722, 2724, 2726, 2728, 2730, 2732, 2734, 2736, 2738, 2740, 2742, 2744,2746, 2748, 2750, 2752, 2754, 2756, 2758, 2760, 2762, 2764, 2766, 2768,2770, 2772, 2774, 2776, 2778, 2780, 2782, 2784, 2786, 2788, 2790, 2792,2794, 2796, 2798, 2800, 2802, 2804, 2806, 2808, 2810, 2812, 2814, 2816,2818, 2820, 2822, 2824, 2826, 2828, 2830, 2832, 2834, 2836, 2838, 2840,2842, 2844, 2846, and 2848, respectively).

Typically, the amino acid sequence of the HHDH polypeptides described inthe above paragraph comprises one or more amino acid residues selectedfrom one of the following subgroups: (1) T at position 2, A or P or S atposition 3, V at position 4, D at position 6, I or F at position 9, L atposition 10, S at position 13, S at position 14, K at position 15, C atposition 16, T or R at position 17, C or S or K at position 20, T atposition 24, Q at position 26, F at position 28, T at position 29, A atposition 30, I at position 31, G at position 33, R at position 34, L atposition 35, N at position 36, H at position 37, D at position 40, L atposition 44, P at position 45, P or A at position 47, N at position 51,V at position 54, R at position 55, D at position 56, K at position 58,G or D at position 61, V at position 63, R at position 72, I at position75, P at position 76, C at position 78, Q at position 80, Y at position82, S or L at position 84, A at position 85, E at position 91, D atposition 93, Q or G at position 95, N at position 96, K at position 107,A at position 112, T or S or G at position 114, A at position 115, P atposition 117, N at position 120, E at position 121, P at position 122, Rat position 126, V at position 130, S at position 133, A or V atposition 134, L or W or V at position 136, H at position 139, I or R atposition 142, S at position 144, S at position 146, T at position 152, Sor A at position 154, V at position 168, T at position 169, F atposition 177, V at position 178, I at position 180, G or I at position181, K at position 184, Y at position 186, L at position 194, N atposition 198, M at position 199, E at position 215, G at position 236, Vat position 237, L at position 238, T at position 240, I or A or V atposition 245, Y at position 249, V or I at position 252, and V atposition 254; or (2) Q at position 38, I at position 52, L at position70, A at position 82, P at position 83, V at position 84, W at position86, R at position 87, D at position 99, T at position 100, A at position174, S at position 189, S at position 195, R at position 203, and T atposition 222; or (3) H at position 37, 1 at position 75, K at position107, A at position 112, A at position 134, A at position 154, V atposition 178, G at position 181, Y at position 186, T at position 222,and V at position 245.

The HHDH polypeptides described in paragraphs 040-046 may have anycombination of two or more, three or more, four or more, five or more,six or more, seven or more, eight or more, nine or more, ten or more,eleven or more, twelve or more, thirteen or more, fourteen or more,fifteen or more, sixteen or more, seventeen more, eighteen or more,nineteen or more, or twenty or more of the above-described amino acidresidues (at the specified positions) in their sequences.

HHDH polypeptides described in paragraphs 040-046 include those encodedby polynucleotides that hybridize to the reference sequence under highlystringent hybridization or under ultra-high stringency conditions.

Nucleic acids “hybridize” when they associate, typically in solution.Nucleic acids hybridize due to a variety of well-characterizedphysico-chemical forces, such as hydrogen bonding, solvent exclusion,base stacking and the like. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) “Laboratory Techniques inbiochemistry and Molecular Biology-Hybridization with Nucleic AcidProbes,” Part I, Chapter 2 (Elsevier, New York).

As used herein, the term “stringent hybridization wash conditions” inthe context of nucleic acid hybridization experiments, such as Southernand Northern hybridizations, are sequence dependent, and are differentunder different environmental parameters. An extensive guide to thehybridization of nucleic acids is found in Tijessen (1993) “LaboratoryTechniques in Biochemistry and Molecular Biology-Hybridization withNucleic Acid Probes,” Part I, Chapter 2 (Elsevier, New York).

For purposes of the present invention, “highly stringent” (or “highstringency”) hybridization and wash conditions are generally selected tobe about 5° C. or less lower than the thermal melting point (T_(m)) forthe specific sequence at a defined ionic strength and pH (as notedbelow, highly stringent conditions can also be referred to incomparative terms). The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of the test sequence hybridizes to aperfectly matched probe. Very stringent conditions are selected to beequal to the T_(m) for a particular probe.

The T_(m) of a nucleic acid duplex indicates the temperature at whichthe duplex is 50% denatured under the given conditions and it representsa direct measure of the stability of the nucleic acid hybrid. Thus, theT_(m) corresponds to the temperature corresponding to the midpoint intransition from helix to random coil; it depends on length, nucleotidecomposition, and ionic strength for long stretches of nucleotides.

After hybridization, unhybridized nucleic acid material can be removedby a series of washes, the stringency of which can be adjusted dependingupon the desired results. Low stringency washing conditions (e.g., usinghigher salt and lower temperature) increase sensitivity, but can producenonspecific hybridization signals and high background signals (i.e.,loses specificity). Higher stringency conditions (e.g., using lower saltand higher temperature that is closer to the hybridization temperature)lowers the background signal, typically with only the specific signalremaining (i.e., increases specificity). See Rapley, R. and Walker, J.M. Eds., “Molecular Biomethods Handbook” (Humana Press, Inc. 1998).

The T_(m) of a DNA-DNA duplex can be estimated using Equation 1 asfollows:T_(m)(° C.)=81.5° C.+16.6(log₁₀M)+0.41(% G+C)−0.72(% f)−500/n,

where M is the molarity of the monovalent cations (usually Na⁺), (% G+C)is the percentage of guanosine (G) and cystosine (C) nucleotides, (% f)is the percentage of formamide and n is the number of nucleotide bases(i.e., length) of the hybrid. See id.

The T_(m) of an RNA-DNA duplex can be estimated by using Equation 2 asfollows: T_(m) (° C.)=79.8° C.+18.5 (log₁₀M)+0.58 (% G+C)−11.8(%G+C)²−0.56 (% f)−820/n, where M is the molarity of the monovalentcations (usually Na+), (% G+C) is the percentage of guanosine (G) andcystosine (C) nucleotides, (% f) is the percentage of formamide and n isthe number of nucleotide bases (i.e., length) of the hybrid. Id.

Equations 1 and 2 are typically accurate only for hybrid duplexes longerthan about 100-200 nucleotides. Id.

The Tm of nucleic acid sequences shorter than 50 nucleotides can becalculated as follows:T_(m)(° C.)=4(G+C)+2(A+T),

where A (adenine), C, T (thymine), and G are the numbers of thecorresponding nucleotides.

An example of stringent hybridization conditions for hybridization ofcomplementary nucleic acids which have more than 100 complementaryresidues on a filter in a Southern or northern blot is 50% formamidewith 1 mg of heparin at 42° C., with the hybridization being carried outovernight. An example of stringent wash conditions is a 0.2×SSC wash at65° C. for 15 minutes (see Sambrook, et al., Molecular Cloning—ALaboratory Manual” (1989) Cold Spring Harbor Laboratory (Cold SpringHarbor, N.Y.) for a description of SSC buffer). Often the highstringency wash is preceded by a low stringency wash to removebackground probe signal. An example low stringency wash is 2×SSC at 40°C. for 15 minutes.

In general, a signal to noise ratio of 2.5×-5× (or higher) than thatobserved for an unrelated probe in the particular hybridization assayindicates detection of a specific hybridization. Detection of at leaststringent hybridization between two sequences in the context of thepresent invention indicates relatively strong structural similarity orhomology to, e.g., the nucleic acids of the present invention providedin the sequence listings herein.

As noted, “highly stringent” conditions are selected to be about 5° C.or less lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH. Target sequences that areclosely related or identical to the nucleotide sequence of interest(e.g., “probe”) can be identified under highly stringent conditions.Lower stringency conditions are appropriate for sequences that are lesscomplementary.

Stringent hybridization (as well as highly stringent, ultra-highstringency, or ultra-ultra high stringency hybridization conditions) andwash conditions can be readily determined empirically for any testnucleic acid. For example, in determining highly stringent hybridizationand wash conditions, the hybridization and wash conditions are graduallyincreased (e.g., by increasing temperature, decreasing saltconcentration, increasing detergent concentration and/or increasing theconcentration of organic solvents, such as formamide, in thehybridization or wash), until a selected set of criteria are met. Forexample, the stringency of hybridization and wash conditions aregradually increased until a probe corresponding to SEQ ID NO: 3, 9, 11,13, 15, 17, 33, 37, 43, 47, 49, 51, 65, 67, 79, 83, 109, 113, 115, 117,153, 157, 161, 163, 165, 169, 179, 191, 199, 261, 263, 265, 269, 421,439, 441, 447, 469, 475, 519, 701, 703, 705, 707, 709, 711, 713, 715,717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743,745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771,773, 775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799,801, 803, 805, 807, 809, 811, 813, 815, 817, 819, 821, 823, 825, 827,829, 831, 833, 835, 837, 839, 841, 843, 845, 847, 849, 851, 853, 855,857, 859, 861, 863, 865, 867, 869, 871, 873, 875, 877, 879, 881, 883,885, 887, 889, 891, 893, 895, 897, 899, 901, 903, 905, 907, 909, 911,913, 915, 917, 919, 921, 923, 925, 927, 929, 931, 933, 935, 937, 939,941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967,969, 971, 973, 975, 979, 981, 983, 985, 987, 989, 991, 993, 995, 997,999, 1001, 1003, 1005, 1007, 1009, 1011, 1013, 1015, 1017, 1019, 1021,1023, 1025, 1027, 1029, 1031, 1033, 1035, 1037, 1039, 1041, 1043, 1045,1047, 1049, 1051, 1053, 1055, 1057, 1059, 1061, 1063, 1065, 1067, 1069,1071, 1073, 1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091, 1093,1095, 1097, 1099, 1101, 1103, 1105, 1107, 1109, 1111, 1113, 1115, 1117,1119, 1121, 1123, 1125, 1127, 1129, 1131, 1133, 1135, 1137, 1139, 1141,1143, 1145, 1147, 1149, 1151, 1153, 1155, 1157, 1159, 1161, 1163, 1165,1167, 1169, 1171, 1173, 1175, 1177, 1179, 1181, 1183, 1185, 1187, 1189,1191, 1193, 1195, 1197, 1199, 1201, 1203, 1205, 1207, 1209, 1211, 1213,1215, 1217, 1219, 1221, 1223, 1225, 1227, 1229, 1231, 1233, 1235, 1237,1239, 1241, 1243, 1245, 1247, 1249, 1251, 1253, 1255, 1257, 1259, 1261,1263, 1265, 1267, 1269, 1271, 1273, 1275, 1277, 1279, 1281, 1283, 1285,1287, 1289, 1291, 1293, 1295, 1297, 1299, 1301, 1303, 1305, 1307, 1309,1311, 1313, 1315, 1317, 1319, 1321, 1323, 1325, 1327, 1329, 1331, 1333,1335, 1337, 1339, 1341, 1343, 1345, 1347, 1349, 1351, 1353, 1355, 1357,1359, 1361, 1363, 1365, 1367, 1369, 1371, 1373, 1375, 1377, 1379, 1381,1383, 1385, 1387, 1389, 1391, 1393, 1395, 1397, 1399, 1401, 1403, 1405,1407, 1409, 1411, 1413, 1415, 1417, 1419, 1421, 1423, 1425, 1427, 1429,1431, 1433, 1435, 1437, 1439, 1441, 1443, 1445, 1447, 1449, 1451, 1453,1455, 1457, 1459, 1461, 1463, 1465, 1467, 1469, 1471, 1473, 1475, 1477,1479, 1481, 1483, 1485, 1487, 1489, 1491, 1493, 1495, 1497, 1499, 1501,1503, 1505, 1507, 1509, 1511, 1513, 1515, 1517, 1519, 1521, 1523, 1525,1527, 1529, 1531, 1533, 1535, 1537, 1539, 1541, 1543, 1545, 1547, 1549,1551, 1553, 1555, 1557, 1559, 1561, 1563, 1565, 1567, 1569, 1571, 1573,1575, 1577, 1579, 1581, 1583, 1585, 1587, 1589, 1591, 1593, 1595, 1597,1599, 1601, 1603, 1605, 1607, 1609, 1611, 1613, 1615, 1617, 1619, 1621,1623, 1625, 1627, 1629, 1631, 1633, 1635, 1637, 1639, 1641, 1643, 1645,1647, 1649, 1651, 1653, 1655, 1657, 1659, 1661, 1663, 1665, 1667, 1669,1671, 1673, 1675, 1677, 1679, 1681, 1683, 1685, 1687, 1689, 1691, 1693,1695, 1697, 1699, 1701, 1703, 1705, 1707, 1709, 1711, 1713, 1715, 1717,1719, 1721, 1723, 1725, 1727, 1729, 1731, 1733, 1735, 1737, 1739, 1741,1743, 1745, 1747, 1749, 1751, 1753, 1755, 1757, 1759, 1761, 1763, 1765,1767, 1769, 1771, 1773, 1775, 1777, 1779, 1781, 1783, 1785, 1787, 1789,1791, 1793, 1795, 1797, 1799, 1801, 1803, 1805, 1807, 1809, 1811, 1813,1815, 1817, 1819, 1821, 1823, 1825, 1827, 1829, 1831, 1833, 1835, 1837,1839, 1841, 1843, 1845, 1847, 1849, 1851, 1853, 1855, 1857, 1859, 1861,1863, 1865, 1867, 1869, 1871, 1873, 1875, 1877, 1879, 1881, 1883, 1885,1887, 1889, 1891, 1893, 1895, 1897, 1899, 1901, 1903, 1905, 1907, 1909,1911, 1913, 1915, 1917, 1919, 1921, 1923, 1925, 1927, 1929, 1931, 1933,1935, 1937, 1939, 1941, 1943, 1945, 1947, 1949, 1951, 1953, 1955, 1957,1959, 1961, 1963, 1965, 1967, 1969, 1971, 1973, 1975, 1977, 1979, 1981,1983, 1985, 1987, 1989, 1991, 1993, 1995, 1997, 1999, 2001, 2003, 2005,2007, 2009, 2011, 2013, 2015, 2017, 2019, 2021, 2023, 2025, 2027, 2029,2031, 2033, 2035, 2037, 2039, 2041, 2043, 2045, 2047, 2049, 2051, 2053,2055, 2057, 2059, 2061, 2063, 2065, 2067, 2069, 2071, 2073, 2075, 2077,2079, 2081, 2083, 2085, 2087, 2089, 2091, 2093, 2095, 2097, 2099, 2101,2103, 2105, 2107, 2109, 2111, 2113, 2115, 2117, 2119, 2121, 2123, 2125,2127, 2129, 2131, 2133, 2135, 2137, 2139, 2141, 2143, 2145, 2147, 2149,2151, 2153, 2155, 2157, 2159, 2161, 2163, 2165, 2167, 2169, 2171, 2173,2175, 2177, 2179, 2181, 2183, 2185, 2187, 2189, 2191, 2193, 2195, 2197,2199, 2201, 2203, 2205, 2207, 2209, 2211, 2213, 2215, 2217, 2219, 2221,2223, 2225, 2227, 2229, 2231, 2233, 2235, 2237, 2239, 2241, 2243, 2245,2247, 2249, 2251, 2253, 2255, 2257, 2259, 2261, 2263, 2265, 2267, 2269,2271, 2273, 2275, 2277, 2279, 2281, 2283, 2285, 2287, 2289, 2291, 2293,2295, 2297, 2299, 2301, 2303, 2305, 2307, 2309, 2311, 2313, 2315, 2317,2319, 2321, 2323, 2325, 2327, 2329, 2331, 2333, 2335, 2337, 2339, 2341,2343, 2345, 2347, 2349, 2351, 2353, 2355, 2357, 2359, 2361, 2363, 2365,2367, 2369, 2371, 2373, 2375, 2377, 2379, 2381, 2383, 2385, 2387, 2389,2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2413,2415, 2417, 2419, 2421, 2423, 2425, 2427, 2429, 2431, 2433, 2435, 2437,2439, 2441, 2443, 2445, 2447, 2449, 2451, 2453, 2455, 2457, 2459, 2461,2463, 2465, 2467, 2469, 2471, 2473, 2475, 2477, 2479, 2481, 2483, 2485,2487, 2489, 2491, 2493, 2495, 2497, 2499, 2501, 2503, 2505, 2507, 2509,2511, 2513, 2515, 2517, 2519, 2521, 2523, 2525, 2527, 2529, 2531, 2533,2535, 2537, 2539, 2541, 2543, 2545, 2547, 2549, 2551, 2553, 2555, 2557,2559, 2561, 2563, 2565, 2567, 2569, 2571, 2573, 2575, 2577, 2579, 2581,2583, 2585, 2587, 2589, 2591, 2593, 2595, 2597, 2599, 2601, 2603, 2605,2607, 2609, 2611, 2613, 2615, 2617, 2619, 2621, 2623, 2625, 2627, 2629,2631, 2633, 2635, 2637, 2639, 2641, 2643, 2645, 2647, 2649, 2651, 2653,2655, 2657, 2659, 2661, 2663, 2665, 2667, 2669, 2671, 2673, 2675, 2677,2679, 2681, 2683, 2685, 2687, 2689, 2691, 2693, 2695, 2697, 2699, 2701,2703, 2705, 2707, 2709, 2711, 2713, 2715, 2717, 2719, 2721, 2723, 2725,2727, 2729, 2731, 2733, 2735, 2737, 2739, 2741, 2743, 2745, 2747, 2749,2751, 2753, 2755, 2757, 2759, 2761, 2763, 2765, 2767, 2769, 2771, 2773,2775, 2777, 2779, 2781, 2783, 2785, 2787, 2789, 2791, 2793, 2795, 2797,2797, 2799, 2801, 2803, 2805, 2807, 2809, 2811, 2813, 2815, 2817, 2819,2821, 2823, 2825, 2827, 2829, 2831, 2833, 2835, 2837, 2839, 2841, 2843,2845, 2847, 2849, 2851, 2853, 2855, 2857, 2859, 2861, or complementarysequence thereof, binds to a perfectly matched complementary target. Atest nucleic acid is said to specifically hybridize to a probe nucleicacid when it hybridizes at least ½ as well to the probe as to theperfectly matched complementary target, i.e., with a signal to noiseratio at least ½ as high as hybridization of the probe to the targetunder conditions in which the perfectly matched probe binds to theperfectly matched complementary target.

Ultra high-stringency hybridization and wash conditions are those inwhich the stringency of hybridization and wash conditions are increaseduntil the signal to noise ratio for binding of the probe to theperfectly matched complementary target nucleic acid is at least 10×. Atarget nucleic acid which hybridizes to a probe under such conditions,with a signal to noise ratio of at least ½ that of the perfectly matchedcomplementary target nucleic acid is said to bind to the probe underultra-high stringency conditions.

Similarly, even higher levels of stringency can be determined bygradually increasing the stringency of hybridization and/or washconditions of the relevant hybridization assay. For example, those inwhich the stringency of hybridization and wash conditions are increaseduntil the signal to noise ratio for binding of the probe to theperfectly matched complementary target nucleic acid is at least 10×,20×, 50×, 100×, or 500×. A target nucleic acid which hybridizes to aprobe under such conditions, with a signal to noise ratio of at least ½that of the perfectly matched complementary target nucleic acid is saidto bind to the probe under ultra-ultra-high stringency conditions.

In one aspect, the present invention provides an isolated or recombinantpolypeptide having HHDH activity and an amino acid sequence that is atleast 80% identical to a reference sequence selected from the groupconsisting of SEQ ID NO: 744, 746, 748, 750, 752, 754, 756, 758, 760,762, 764, 766, 768, 770, 772, 774, 776, 778, 780, 782, 784, 786, 788,790, 792, 794, 796, 798, 800, 802, 804, 806, 808, 810, 812, 814, 816,818, 820, 822, 824, 826, 828, 830, 832, 834, 836, 838, 840, 842, 844,846, 848, 850, 852, 854, 856, 858, 860, 862, 864, 866, 868, 870, 872,874, 876, 878, 880, 882, 884, 886, 888, 890, 892, 894, 896, 898, 900,902, 904, 906, 908, 910, 912, 914, 916, 918, 920, 922, 924, 926, 928,930, 932, 934, 936, 938, 940, 942, 944, 946, 948, 950, 952, 954, 956,958, 960, 962, 964, 966, 968, 970, 972, 974, 976, 978, 980, 982, 984,986, 988, 990, 992, 994, 996, 998, 1000, 1002, 1004, 1006, 1008, 1010,1012, 1014, 1016, 1018, 1020, 1022, 1024, 1026, 1028, 1030, 1032, 1034,1036, 1038, 1040, 1042, 1044, 1046, 1048, 1050, 1052, 1054, 1056, 1058,1060, 1062, 1064, 1066, 1068, 1070, 1072, 1074, 1076, 1078, 1080, 1082,1084, 1086, 1088, 1090, 1092, 1094, 1096, 1098, 1100, 1102, 1104, 1106,1108, 1110, 1112, 1114, 1116, 1118, 1120, 1122, 1124, 1126, 1128, 1130,1132, 1134, 1136, 1138, 1140, 1142, 1144, 1146, 1148, 1150, 1152, 1154,1156, 1158, 1160, 1162, 1164, 1166, 1168, 1170, 1172, 1174, 1176, 1178,1180, 1182, 1184, 1186, 1188, 1190, 1192, 1194, 1196, 1198, 1110, 1112,1114, 1116, 1118, 1120, 1122, 1124, 1126, 1128, 1130, 1132, 1134, 1136,1138, 1140, 1142, 1144, 1146, 1148, 1150, 1152, 1154, 1156, 1158, 1160,1162, 1164, 1166, 1168, 1170, 1172, 1174, 1176, 1178, 1180, 1182, 1184,1186, 1188, 1190, 1192, 1194, 1196, 1198, 1200, 1202, 1204, 1206, 1208,1210, 1212, 1214, 1216, 1218, 1220, 1222, 1224, 1226, 1228, 1230, 1232,1234, 1236, 1238, 1240, 1242, 1244, 1246, 1248, 1250, 1252, 1254, 1256,1258, 1260, 1262, 1264, 1266, 1268, 1270, 1272, 1274, 1276, 1278, 1280,1282, 1284, 1286, 1288, 1290, 1292, 1294, 1296, 1298, 1300, 1302, 1304,1306, 1308, 1310, 1312, 1314, 1316, 1318, 1320, 1322, 1324, 1326, 1328,1330, 1332, 1334, 1336, 1338, 1340, 1342, 1344, 1346, 1348, 1350, 1352,1354, 1356. 1358. 1360, 1362, 1364, 1366, 1368, 1370, 1372, 1374, 1376,1378, 1380, 1382, 1384, 1386, 1388, 1390, 1392, 1394, 1396, 1398, 1400,1402, 1404, 1406, 1408, 1410, 1412, 1414, 1416, 1418, 1420, 1422, 1424,1426, 1428, 1430, 1432, 1434, 1436, 1438, 1440, 1442, 1444, 1446, 1448,1450, 1452, 1454, 1456, 1458, 1460, 1462, 1464, 1468, 1470, 1472, 1474,1476, 1478, 1480, 1482, 1484, 1486, 1488, 1490, 1492, 1494, 1496, 1498,1500, 1502, 1504, 1506, 1508, 1510, 1512, 1514, 1516, 1518, 1520, 1522,1524, 1526, 1528, 1530, 1532, 1534, 1536, 1538, 1540, 1542, 1544, 1546,1548, 1550, 1552, 1554, 1556, 1558, 1560, 1562, 1564, 1566, 1568, 1570,1572, 1574, 1576, 1578, 1580, 1582, 1584, 1586, 1588, 1590, 1592, 1594,1596, 1598, 1600, 1602, 1604, 1606, 1608, 1610, 1612, 1614, 1616, 1618,1620, 1622, 1624, 1626, 1628, 1630, 1632, 1634, 1636, 1638, 1640, 1642,1644, 1646, 1648, 1650, 1652, 1654, 1656, 1658, 1660, 1662, 1664, 1666,1668, 1670, 1672, 1674, 1676, 1678, 1680, 1682, 1684, 1684, 1686, 1688,1690, 1692, 1694, 1696, 1698, 1700, 1702, 1704, 1706, 1708, 1710, 1712,1714, 1716, 1718, 1720, 1722, 1724, 1726, 1728, 1730, 1732, 1734, 1736,1738, 1740, 1742, 1744, 1746, 1748, 1750, 1752, 1754, 1756, 1758, 1760,1762, 1764, 1766, 1768, 1770, 1772, 1774, 1776, 1778, 1780, 1782, 1784,1786, 1788, 1790, 1792, 1794, 1796, 1798, 1800, 1802, 1804, 1806, 1808,1810, 1812, 1814, 1816, 1818, 1820, 1822, 1824, 1826, 1828, 1830, 1832,1834, 1836, 1838, 1840, 1842, 1844, 1846, 1848, 1850, 1852, 1854, 1856,1858, 1860, 1862, 1864, 1866, 1868, 1870, 1872, 1874, 1876, 1878, 1880,1882, 1884, 1886, 1888, 1890, 1892, 1894, 1896, 1898, 1900, 1902, 1904,1906, 1908, 1910, 1912, 1914, 1916, 1918, 1920, 1922, 1924, 1926, 1928,1930, 1932, 1934, 1936, 1938, 1940, 1942, 1944, 1946, 1948, 1950, 1952,1954, 1956, 1958, 1960, 1962, 1964, 1966, 1968, 1970, 1972, 1974, 1976,1978, 1980, 1982, 1984, 1986, 1988, 1990, 1992, 1994, 1996, 1998, 2000,2002, 2004, 2006, 2008, 2010, 2012, 2014, 2016, 2018, 2020, 2022, 2024,2026, 2028, 2030, 2032, 2034, 2036, 2038, 2040, 2042, 2044, 2046, 2048,2050, 2052, 2054, 2056, 2058, 2060, 2062, 2064, 2066, 2068, 2070, 2072,2074, 2076, 2078, 2080, 2082, 2084, 2086, 2088, 2090, 2092, 2094, 2096,2098, 2100, 2102, 2104, 2106, 2108, 2110, 2112, 2114, 2116, 2118, 2120,2122, 2124, 2126, 2128, 2130, 2132, 2134, 2136, 2138, 2140, 2142, 2144,2146, 2148, 2150, 2152, 2154, 2156, 2158, 2160, 2162, 2164, 2166, 2168,2170, 2172, 2174, 2176, 2178, 2180, 2182, 2184, 2186, 2188, 2190, 2192,2194, 2196, 2198, 2200, 2202, 2204, 2206, 2208, 2210, 2212, 2214, 2216,2218, 2220, 2222, 2224, 2226, 2228, 2230, 2232, 2234, 2236, 2238, 2240,2242, 2244, 2246, 2248, 2250, 2252, 2254, 2256, 2258, 2260, 2262, 2264,2266, 2268, 2270, 2272, 2274, 2276, 2278, 2280, 2282, 2284, 2286, 2288,2290, 2292, 2294, 2296, 2298, 2300, 2302, 2304, 2306, 2308, 2310, 2312,2314, 2316, 2318, 2320, 2322, 2324, 2326, 2328, 2330, 2332, 2334, 2336,2338, 2340, 2342, 2344, 2346, 2348, 2350, 2352, 2354, 2356, 2358, 2360,2362, 2364, 2366, 2368, 2370, 2372, 2374, 2376, 2378, 2380, 2382, 2384,2386, 2388, 2390, 2392, 2394, 2396, 2398, 2400, 2402, 2404, 2406, 2408,2410, 2412, 2414, 2416, 2418, 2420, 2422, 2424, 2426, 2428, 2430, 2432,2434, 2436, 2438, 2440, 2442, 2444, 2446, 2448, 2450, 2452, 2454, 2456,2458, 2460, 2462, 2464, 2466, 2468, 2470, 2472, 2474, 2476, 2478, 2480,2482, 2484, 2486, 2488, 2490, 2492, 2494, 2496, 2498, 2500, 2502, 2504,2506, 2508, 2510, 2512, 2514, 2516, 2518, 2520, 2522, 2524, 2526, 2528,2530, 2532, 2534, 2526, 2528, 2530, 2532, 2534, 2536, 2538, 2540, 2542,2544, 2546, 2548, 2550, 2552, 2554, 2556, 2558, 2560, 2562, 2564, 2566,2568, 2570, 2572, 2574, 2576, 2578, 2580, 2582, 2584, 2586, 2588, 2590,2592, 2594, 2596, 2598, 2600, 2602, 2604, 2606, 2608, 2610, 2612, 2614,2616, 2618, 2620, 2622, 2624, 2626, 2628, 2630, 2632, 2634, 2636, 2638,2640, 2642, 2644, 2646, 2648, 2650, 2652, 2654, 2656, 2658, 2660, 2662,2664, 2668, 2670, 2672, 2674, 2676, 2678, 2680, 2682, 2684, 2686, 2688,2690, 2692, 2694, 2696, 2698, 2700, 2702, 2704, 2706, 2708, 2710, 2712,2714, 2716, 2718, 2720, 2722, 2724, 2726, 2728, 2730, 2732, 2734, 2736,2738, 2740, 2742, 2744, 2746, 2748, 2750, 2752, 2754, 2756, 2758, 2760,2762, 2764, 2766, 2768, 2770, 2772, 2774, 2776, 2778, 2780, 2782, 2784,2786, 2788, 2790, 2792, 2794, 2796, 2798, 2800, 2802, 2804, 2806, 2808,2810, 2812, 2814, 2816, 2818, 2820, 2822, 2824, 2826, 2828, 2830, 2832,2834, 2836, 2838, 2840, 2842, 2844, 2846, and 2848. Certain of theseHHDH polypeptides may be at least 81% identical, 82% identical, 83%identical, 84% identical, 85% identical, 86% identical, 87% identical,88% identical, 89% identical, 90% identical, 91% identical, 92%identical, 93% identical, 94% identical, 95% identical, 96% identical,97% identical, 98% or 99% identical to the reference sequences.

Typically, HHDH polypeptides described in paragraph 064 include thosehaving an amino acid sequence that comprises one or more amino acidresidues selected from the group consisting of V at position 5, A atposition 27, V at position 45, Q at position 46, I at position 54, V atposition 60, V at position 65, N at position 67, H at position 72, I atposition 77, S at position 87, R at position 91, T at position 93, A atposition 95, E at position 96, M or Q at position 100, T at position103, R or T at position 117, S at position 118, R or I or L or Y atposition 121, S at position 135, R at position 139+E at position 176 orD at position 139+R at position 176, A at position 146, N at position153, H at position 166, G or A at position 177, M or C at position 178,N or D at position 179, D or T at position 180, K at position 181, N atposition 182, V at position 190, A at position 199, W or Y at position201, Y at position 205, L at position 236, T or R at position 238, F atposition 239, V at position 246, E or A or S at position 251, and P atposition 252, wherein amino acid position is determined by optimalalignment of the amino acid sequence of the HHDH polypeptide with thereference sequence.

In some embodiments, the above-described HHDH polypeptides are encodedby an amino acid sequence that comprises one or more amino acid residuesselected from the subgroup consisting of A at position 27, Q at position46, I at position 54, V at position 60, V at position 65, S at position87, R at position 91, A at position 95, E at position 96, M or Q atposition 100, R at position 117, S at position 118, R at position 121, Sat position 135, A at position 146, N at position 153, M at position178, N at position 179, N at position 182, V at position 190, A atposition 199, Y at position 201, Y at position 205, L at position 236, Tat position 238, and E or A at position 251. In certain embodiments,these HHDH polypeptides are encoded by an amino acid sequence having oneor more amino acid residues selected from the subgroup consisting of Vat position 5, V at position 45, N at position 67, H at position 72, Iat position 77, T at position 93, T at position 103, T at position 117,S at position 118, I or L or Y at position 121, R at position 139+E atposition 176 or D at position 139+R at position 176, H at position 166,G or A at position 177, C at position 178, D at position 179, W atposition 201, R at position 238, F at position 239, V at position 246, Sat position 251, and P at position 252. In other embodiments, these HHDHpolypeptides are encoded by an amino acid sequence that comprises one ormore residues selected from the group consisting of I at position 54, Vat position 60, N at position 67, H at position 72, T at position 93, Mat position 100, T at position 117, R at position 121, S at position135, A at position 146, A or G at position 177, M or C at position 178,D at position 179, W at position 201, Y at position 205, R at position238, F at position 239, and V at position 246. Some of these HHDHpolypeptides have an amino acid sequence that comprises one or moreamino acid residues selected from the subgroup group consisting of N atposition 67, H at position 72, T at position 117, R at position 121, Sat position 135, A at position 146, A at position 177, D at position179, W at position 201, Y at position 205, and V at position 246. HHDHpolypeptides of the present invention include those having an amino acidsequence comprising the following combination of amino acid residues: Nat position 67+H at position 72+T at position 117+R at position 121+S atposition 135+A at position 146+A at position 154+A at position 177+D atposition 179+W at position 201+Y at position 205+V at position 246. Inother embodiments the HHDH polypeptides have an amino acid sequence thatcomprises one or more amino acid residues selected from the groupconsisting of I at position 54, V at position 60, E at position 91, T atposition 93, M at position 100, G at position 177, M or C at position178, R at position 238, and F at position 239.

HHDH polypeptides described in paragraph 064 may have an amino acidsequence that comprises one or more amino acid residues selected fromthe group consisting of T at position 2, A or P or S at position 3, V atposition 4, D at position 6, I or F at position 9, L at position 10, Sat position 13, S at position 14, K at position 15, C at position 16, Tor R at position 17, C or S or K at position 20, T at position 24, Q atposition 26, F at position 28, T at position 29, A at position 30, I atposition 31, G at position 33, R at position 34, L at position 35, N atposition 36, H at position 37, Q at position 38, D at position 40, L atposition 44, P at position 45, P or A at position 47, N at position 51,I at position 52, V at position 54, R at position 55, D at position 56,K at position 58, G or D at position 61, V at position 63, L at position70, R at position 72, I at position 75, P at position 76, C at position78, Q at position 80, A or Y at position 82, P at position 83, S or L orV at position 84, A at position 85, W at position 86, R at position 87,E at position 91, D at position 93, Q or G at position 95, N at position96, D at position 99, T at position 100, K at position 107, A atposition 112, T or S or G at position 114, A at position 115, P atposition 117, N at position 120, E at position 121, P at position 122, Rat position 126, V at position 130, S at position 133, A or V atposition 134, L or W or V at position 136, H at position 139, I or R atposition 142, S at position 144, S at position 146, T at position 152, Sor A at position 154, V at position 168, T at position 169, A atposition 174, F at position 177, V at position 178, I at position 180, Gor I at position 181, K at position 184, Y at position 186, S atposition 189, L at position 194, S at position 195, N at position 198, Mat position 199, R at position 203, E at position 215, T at position222, G at position 236, V at position 237, L at position 238, T atposition 240, I or A or V at position 245, Y at position 249, V or I atposition 252, and V at position 254.

In addition to the amino acid residues described in paragraphs 065-066,the specified HHDH polypeptides may further comprise one or moreadditional amino acid residues selected from the group consisting of Tat position 2, A or P or S at position 3, V at position 4, D at position6, I or F at position 9, L at position 10, S at position 13, S atposition 14, K at position 15, C at position 16, T or R at position 17,C or S or K at position 20, T at position 24, Q at position 26, F atposition 28, T at position 29, A at position 30, I at position 31, G atposition 33, R at position 34, L at position 35, N at position 36, H atposition 37, Q at position 38, D at position 40, L at position 44, P atposition 45 (if not V from paragraphs 065-066), P or A at position 47, Nat position 51, I at position 52, V at position 54 (if not I), R atposition 55, D at position 56, K at position 58, G or D at position 61,V at position 63, L at position 70, R at position 72 (if not H), I atposition 75, P at position 76, C at position 78, Q at position 80, A orY at position 82, P at position 83, S or L or V at position 84, A atposition 85, W at position 86, R at position 87, E at position 91 (ifnot R), D at position 93 (if not T), Q or G at position 95 (if not A), Nat position 96 (if not E), D at position 99, T at position 100, K atposition 107, A at position 112, T or S or G at position 114, A atposition 115, P at position 117 (if not R or T), N at position 120, E atposition 121 (if not R or I or L or Y), P at position 122, R at position126, V at position 130, S at position 133, A or V at position 134, L orW or V at position 136, H at position 139 (if not R or D), I or R atposition 142, S at position 144, S at position 146 (if not A), T atposition 152, S or A at position 154, V at position 168, T at position169, A at position 174, F at position 177 (if not G or A), V at position178 (if not M or C), I at position 180, G or I at position 181, K atposition 184, Y at position 186, S at position 189, L at position 194, Sat position 195, N at position 198, M at position 199 (if not A), R atposition 203, E at position 215, T at position 222, G at position 236(if not L), V at position 237, L at position 238 (if not T or R), T atposition 240, 1 or A or V at position 245, Y at position 249, V or I atposition 252 (if not P), and V at position 254, wherein amino acidresidue position is determined by optimal alignment of the amino acidsequence of the HHDH polypeptide with the reference sequence. Some ofthese HHDH polypeptides have an amino acid sequence that furthercomprises one or more amino acid residues selected from the subgroupconsisting of H at position 37, Q at position 38, I at position 52, L atposition 70, I at position 75, A at position 82, P at position 83, V atposition 84, W at position 86, R at position 87, D at position 99, T atposition 100, K at position 107, A at position 112, A at position 134, Aat position 154, A at position 174, V at position 178 (if not M or C), Gat position 181, Y at position 186, S at position 189, S at position195, R at position 203, T at position 222, and V at position 245.

Some of the HHDH polypeptides described in paragraphs 065-066 furthercomprise one or more amino acid residues selected from one of thefollowing subgroups: (1) T at position 2, A or P or S at position 3, Vat position 4, D at position 6, I or F at position 9, L at position 10,S at position 13, S at position 14, K at position 15, C at position 16,T or R at position 17, C or S or K at position 20, T at position 24, Qat position 26, F at position 28, T at position 29, A at position 30, Iat position 31, G at position 33, R at position 34, L at position 35, Nat position 36, H at position 37, D at position 40, L at position 44, Pat position 45 (if not I), P or A at position 47, N at position 51, V atposition 54 (if not I), R at position 55, D at position 56, K atposition 58, G or D at position 61, V at position 63, R at position 72(if not H), I at position 75, P at position 76, C at position 78, Q atposition 80, Y at position 82, S or L at position 84, A at position 85,E at position 91 (if not R), D at position 93 (if not T), Q or G atposition 95 (if not A), N at position 96 (if not E), K at position 107,A at position 112, T or S or G at position 114, A at position 115, P atposition 117 (if not R or T), N at position 120, E at position 121 (ifnot R or I or L or Y), P at position 122, R at position 126, V atposition 130, S at position 133, A or V at position 134, L or W or V atposition 136, H at position 139 (if not R or D), I or R at position 142,S at position 144, S at position 146 (if not A), T at position 152, S orA at position 154, V at position 168, T at position 169, F at position177 (if not G or A), V at position 178 (if not M or C), I at position180, G or I at position 181, K at position 184, Y at position 186, L atposition 194, N at position 198, M at position 199 (if not A), E atposition 215, G at position 236, V at position 237, L at position 238(if not T or R), T at position 240, I or A or V at position 245, Y atposition 249, V or I at position 252 (if not P), and V at position 254;or (2) Q at position 38, I at position 52, L at position 70, A atposition 82, P at position 83, V at position 84, W at position 86, R atposition 87, D at position 99, T at position 100, A at position 174, Sat position 189, S at position 195, R at position 203, and T at position222; or (3) H at position 37, I at position 75, K at position 107, A atposition 112, A at position 134, A at position 154, V at position 178(if not M or C), G at position 181, Y at position 186, T at position222, and V at position 245. The parentheticals in the above paragraphs(“if not X”) refers to the proviso in which the specified residue isexcluded from the specified amino acid residue position if amino acidresidue X is selected from paragraphs 065-066 for that residue position.

The HHDH polypeptides of paragraphs 065-069 may have any combination oftwo or more, three or more, four or more, five or more, six or more,seven or more, eight or more, nine or more, ten or more, eleven or more,twelve or more, thirteen or more, fourteen or more, fifteen or more,sixteen or more, seventeen more, eighteen or more, nineteen or more, ortwenty or more of the above-described amino acid residues (at thespecified positions) in their sequences.

In a specific embodiment, the isolated or recombinant polypeptide havingHHDH activity is a polypeptide selected from the group consisting of:(a) a polypeptide comprising an amino acid sequence that is at least 86%identical (sometimes at least 87% identical, 88% identical, 89%identical, 90% identical, 91% identical, 92% identical, 93% identical,94% identical, 95% identical, 96% identical, 97% or 98% identical) to areference sequence selected from the group consisting of SEQ ID NO: 750and SEQ ID NO: 2848; (b) a polypeptide comprising an amino acid sequencethat is at least 93% identical (sometimes at least 94% identical, 95%identical, 96% identical, 97% identical, 98% or 99% identical) to areference sequence selected from the group consisting of SEQ ID NO: 200and SEQ ID NO: 1978; (c) a polypeptide comprising an amino acid sequencethat is at least 94% identical (sometimes at least 95% identical, 96%identical, 97% identical, 98% or 99% identical) to reference sequenceSEQ ID NO: 2496; (d) a polypeptide comprising an amino acid sequencethat is at least 95% identical (sometimes at least 96% identical, 97%identical, 98% or 99% identical) to a reference sequence selected fromthe group consisting of SEQ ID NO: 264, SEQ ID NO: 266, SEQ ID NO: 470,SEQ ID NO: 476, SEQ ID NO: 2846, SEQ ID NO: 2130, SEQ ID NO: 1926, SEQID NO: 2348, SEQ ID NO: 1984, and SEQ ID NO: 2446; (e) a polypeptidecomprising an amino acid sequence that is at least 96% identical(sometimes at least 97% identical, 98% or 99% identical) to a referencesequence selected from the group consisting of SEQ ID NO: 116, SEQ IDNO: 448, SEQ ID NO: 2834, SEQ ID NO: 1820, SEQ ID NO: 2040, and SEQ IDNO: 2838; (f) a polypeptide comprising an amino acid sequence that is atleast 97% identical (sometimes at least 98% or 99% identical) to areference sequence selected from the group consisting of SEQ ID NO: 110,SEQ ID NO: 162, SEQ ID NO: 262, SEQ ID NO: 422, SEQ ID NO: 440, SEQ IDNO: 520, SEQ ID NO: 1276, SEQ ID NO: 1710, SEQ ID NO: 1824, SEQ ID NO:2582, SEQ ID NO: 1818, SEQ ID NO: 1164, SEQ ID NO: 1208, SEQ ID NO:1750, and SEQ ID NO: 2808; (g) a polypeptide comprising an amino acidsequence that is at least 98% identical (sometimes at least 99%identical) to a reference sequence selected from the group consisting ofSEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 68, SEQ ID NO: 118, SEQ ID NO:164, SEQ ID NO: 166, SEQ ID NO: 180, SEQ ID NO: 1826, SEQ ID NO: 2598,SEQ ID NO: 1324, SEQ ID NO: 1898, SEQ ID NO: 2146, SEQ ID NO: 1762, SEQID NO: 2580, SEQ ID NO: 2724, SEQ ID NO: 1698, SEQ ID NO: 1648, SEQ IDNO: 2576, SEQ ID NO: 2790, and SEQ ID NO: 1760; and (h) a polypeptidecomprising an amino acid sequence that is at least 99% identical to areference sequence selected from the group consisting of SEQ ID NO: 4,SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 34, SEQ ID NO:38, SEQ ID NO: 44, SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 66, SEQ IDNO: 80, SEQ ID NO: 84, SEQ ID NO: 114, SEQ ID NO: 154, SEQ ID NO: 158,SEQ ID NO: 170, SEQ ID NO: 264, SEQ ID NO: 266, SEQ ID NO: 270, SEQ IDNO: 470, SEQ ID NO: 476, SEQ ID NO: 1858, SEQ ID NO: 1212, SEQ ID NO:2566, SEQ ID NO: 1388, SEQ ID NO: 1316, SEQ ID NO: 1058, SEQ ID NO:1506, SEQ ID NO: 1266, SEQ ID NO: 1616, SEQ ID NO: 1292, SEQ ID NO:1770, SEQ ID NO: 1758, SEQ ID NO: 1656, SEQ ID NO: 1800, SEQ ID NO:1200, SEQ ID NO: 1354, SEQ ID NO: 1248, SEQ ID NO: 2594, SEQ ID NO:1508, SEQ ID NO: 2680, SEQ ID NO: 1904, SEQ ID NO: 1364, SEQ ID NO:1580, SEQ ID NO: 2214, SEQ ID NO: 2710, SEQ ID NO: 1670, SEQ ID NO:1286, SEQ ID NO: 2784, SEQ ID NO: 2782, SEQ ID NO: 1174, SEQ ID NO:1618, SEQ ID NO: 970, SEQ ID NO: 1214, SEQ ID NO: 1780, SEQ ID NO: 1452,SEQ ID NO: 1766, and SEQ ID NO: 1156.

The present invention also provides a polypeptide, typically an isolatedor recombinant polypeptide having HHDH activity greater than thewild-type HHDH of SEQ ID NO: 2, and having an amino acid sequence thatis at least 89% identical to SEQ ID NO: 442; typically, at least 93%identical to SEQ ID NO: 442; more typically, at least 95% identical toSEQ ID NO: 442, sometimes at least 97% identical to SEQ ID NO: 442;optionally, at least 99% identical to SEQ ID NO: 442.

In another embodiment, the present invention is directed to apolypeptide, typically an isolated or recombinant polypeptide havingHHDH activity greater than the wild-type HHDH of SEQ ID NO: 2, andhaving an amino acid sequence that is at least 88% identical to SEQ IDNO: 702; typically, at least 93% identical to SEQ ID NO: 702; moretypically, at least 95% identical to SEQ ID NO: 702; sometimes, at least97% identical to SEQ ID NO: 702; optionally, at least 99% identical toSEQ ID NO: 702.

In a further embodiment, the present invention provides an HHDHpolypeptide having an amino acid sequence that is at least 96% identicalto SEQ ID NO: 116 or 448. HHDH polypeptides of the present inventioninclude those that are at least 97% identical, 98% or 99% identical toSEQ ID NO: 116 or 448.

The present invention further provides an HHDH polypeptide having anamino acid sequence that is at least 95% identical to SEQ ID NO: 264,266, 470 or 476. Desirable HHDH polypeptides of the present inventioninclude those that are at least 96% identical, 97% identical, 98% or 99%identical to SEQ ID NO: 264, 266, 470 or 476.

In some embodiments, the HHDH polypeptides described in paragraphs071-075 have an amino acid sequence that comprises one or more aminoacid residues selected from the group consisting of V at position 5, Aat position 27, V at position 45, Q at position 46, I at position 54, Vat position 60, V at position 65, N at position 67, H at position 72, Iat position 77, S at position 87, R at position 91, T at position 93, Aat position 95, E at position 96, M or Q at position 100, T at position103, R or T at position 117, S at position 118, R or I or L or Y atposition 121, S at position 135, R at position 139+E at position 176 orD at position 139+R at position 176, A at position 146, N at position153, H at position 166, G or A at position 177, M or C at position 178,N or D at position 179, D or T at position 180, K at position 181, N atposition 182, Vat position 190, A at position 199, W or Y at position201, Y at position 205, L at position 236, T or R at position 238, F atposition 239, V at position 246, E or A or S at position 251, and P atposition 252, wherein amino acid position is determined by optimalalignment of the amino acid sequence with the reference sequence. Insome embodiments, the above-described HHDH polypeptides are encoded byan amino acid sequence that comprises one or more amino acid residuesselected from the subgroup consisting of A at position 27, Q at position46, I at position 54, V at position 60, V at position 65, S at position87, R at position 91, A at position 95, E at position 96, M or Q atposition 100, R at position 117, S at position 118, R at position 121, Sat position 135, A at position 146, N at position 153, M at position178, N at position 179, N at position 182, V at position 190, A atposition 199, Y at position 201, Y at position 205, L at position 236, Tat position 238, and E or A at position 251.

In certain embodiments, these HHDH polypeptides are encoded by an aminoacid sequence having one or more amino acid residues selected from thesubgroup consisting of V at position 5, V at position 45, N at position67, H at position 72, I at position 77, T at position 93, T at position103, T at position 117, S at position 118, I or L or Y at position 121,R at position 139+E at position 176 or D at position 139+R at position176, H at position 166, G or A at position 177, C at position 178, D atposition 179, W at position 201, R at position 238, F at position 239, Vat position 246, S at position 251, and P at position 252. In otherembodiments, these HHDH polypeptides are encoded by an amino acidsequence that comprises one or more residues selected from the groupconsisting of I at position 54, V at position 60, N at position 67, H atposition 72, T at position 93, M at position 100, T at position 117, Rat position 121, S at position 135, A at position 146, A or G atposition 177, M or C at position 178, D at position 179, W at position201, Y at position 205, R at position 238, F at position 239, and V atposition 246. Some of these HHDH polypeptides have an amino acidsequence that comprises one or more amino acid residues selected fromthe subgroup group consisting of N at position 67, H at position 72, Tat position 117, R at position 121, S at position 135, A at position146, A at position 177, D at position 179, W at position 201, Y atposition 205, and V at position 246. HHDH polypeptides of the presentinvention include those having an amino acid sequence comprising thefollowing combination of amino acid residues: N at position 67+H atposition 72+T at position 117+R at position 121+S at position 135+A atposition 146+A at position 154+A at position 177+D at position 179+W atposition 201+Y at position 205+V at position 246. In other embodimentsthe HHDH polypeptides have an amino acid sequence that comprises one ormore amino acid residues selected from the group consisting of I atposition 54, V at position 60, E at position 91, T at position 93, M atposition 100, G at position 177, M or C at position 178, R at position238, and F at position 239.

HHDH polypeptides described in paragraphs 071-075 may have an amino acidsequence that comprises one or more amino acid residues selected fromthe group consisting of T at position 2, A or P or S at position 3, V atposition 4, D at position 6, I or F at position 9, L at position 10, Sat position 13, S at position 14, K at position 15, C at position 16, Tor R at position 17, C or S or K at position 20, T at position 24, Q atposition 26, F at position 28, T at position 29, A at position 30, I atposition 31, G at position 33, R at position 34, L at position 35, N atposition 36, H at position 37, Q at position 38, D at position 40, L atposition 44, P at position 45, P or A at position 47, N at position 51,I at position 52, V at position 54, R at position 55, D at position 56,K at position 58, G or D at position 61, V at position 63, L at position70, R at position 72, I at position 75, P at position 76, C at position78, Q at position 80, A or Y at position 82, P at position 83, S or L orV at position 84, A at position 85, W at position 86, R at position 87,E at position 91, D at position 93, Q or G at position 95, N at position96, D at position 99, T at position 100, K at position 107, A atposition 112, T or S or G at position 114, A at position 115, P atposition 117, N at position 120, E at position 121, P at position 122, Rat position 126, V at position 130, S at position 133, A or V atposition 134, L or W or V at position 136, H at position 139, I or R atposition 142, S at position 144, S at position 146, T at position 152, Sor A at position 154, V at position 168, T at position 169, A atposition 174, F at position 177, V at position 178, I at position 180, Gor I at position 181, K at position 184, Y at position 186, S atposition 189, L at position 194, S at position 195, N at position 198, Mat position 199, R at position 203, E at position 215, T at position222, G at position 236, V at position 237, L at position 238, T atposition 240, I or A or V at position 245, Y at position 249, V or I atposition 252, and V at position 254.

In addition to the amino acid residues described above, the HHDHpolypeptides described in paragraphs 076-077 may further comprise one ormore additional amino acid residues selected from the group consistingof T at position 2, A or P or S at position 3, V at position 4, D atposition 6, I or F at position 9, L at position 10, S at position 13, Sat position 14, K at position 15, C at position 16, T or R at position17, C or S or K at position 20, T at position 24, Q at position 26, F atposition 28, T at position 29, A at position 30, I at position 31, G atposition 33, R at position 34, L at position 35, N at position 36, H atposition 37, Q at position 38, D at position 40, L at position 44, P atposition 45 (if not V from paragraphs 076-077), P or A at position 47, Nat position 51, I at position 52, V at position 54 (if not I), R atposition 55, D at position 56, K at position 58, G or D at position 61,V at position 63, L at position 70, R at position 72 (if not H), I atposition 75, P at position 76, C at position 78, Q at position 80, A orY at position 82, P at position 83, S or L or V at position 84, A atposition 85, W at position 86, R at position 87, E at position 91 (ifnot R), D at position 93 (if not T), Q or G at position 95 (if not A), Nat position 96 (if not E), D at position 99, T at position 100, K atposition 107, A at position 112, T or S or G at position 114, A atposition 115, P at position 117 (if not R or T), N at position 120, E atposition 121 (if not R or I or L or Y), P at position 122, R at position126, V at position 130, S at position 133, A or V at position 134, L orW or V at position 136, H at position 139 (if not R or D), I or R atposition 142, S at position 144, S at position 146 (if not A), T atposition 152, S or A at position 154, V at position 168, T at position169, A at position 174, F at position 177 (if not G or A), V at position178 (if not M or C), I at position 180, G or I at position 181, K atposition 184, Y at position 186, S at position 189, L at position 194, Sat position 195, N at position 198, M at position 199 (if not A), R atposition 203, E at position 215, T at position 222, G at position 236(if not L), V at position 237, L at position 238 (if not T or R), T atposition 240, I or A or V at position 245, Y at position 249, V or I atposition 252 (if not P), and V at position 254, wherein amino acidresidue position is determined by optimal alignment of the amino acidsequence of the HHDH polypeptide with the reference sequence. Some ofthese HHDH polypeptides have an amino acid sequence that furthercomprises one or more amino acid residues selected from the subgroupconsisting of H at position 37, Q at position 38, I at position 52, L atposition 70, I at position 75, A at position 82, P at position 83, V atposition 84, W at position 86, R at position 87, D at position 99, T atposition 100, K at position 107, A at position 112, A at position 134, Aat position 154, A at position 174, V at position 178 (if not M or C), Gat position 181, Y at position 186, S at position 189, S at position195, R at position 203, T at position 222, and V at position 245.

Some of the HHDH polypeptides of paragraphs 076-077 further comprise oneor more amino acid residues selected from one of the followingsubgroups: (1) T at position 2, A or P or S at position 3, V at position4, D at position 6, I or F at position 9, L at position 10, S atposition 13, S at position 14, K at position 15, C at position 16, T orR at position 17, C or S or K at position 20, T at position 24, Q atposition 26, F at position 28, T at position 29, A at position 30, I atposition 31, G at position 33, R at position 34, L at position 35, N atposition 36, H at position 37, D at position 40, L at position 44, P atposition 45 (if not I), P or A at position 47, N at position 51, V atposition 54 (if not I), R at position 55, D at position 56, K atposition 58, G or D at position 61, V at position 63, R at position 72(if not H), I at position 75, P at position 76, C at position 78, Q atposition 80, Y at position 82, S or L at position 84, A at position 85,E at position 91 (if not R), D at position 93 (if not T), Q or G atposition 95 (if not A), N at position 96 (if not E), K at position 107,A at position 112, T or S or G at position 114, A at position 115, P atposition 117 (if not R or T), N at position 120, E at position 121 (ifnot R or I or L or Y), P at position 122, R at position 126, V atposition 130, S at position 133, A or V at position 134, L or W or V atposition 136, H at position 139 (if not R or D), I or R at position 142,S at position 144, S at position 146 (if not A), T at position 152, S orA at position 154, V at position 168, T at position 169, F at position177 (if not G or A), V at position 178 (if not M or C), I at position180, G or I at position 181, K at position 184, Y at position 186, L atposition 194, N at position 198, M at position 199 (if not A), E atposition 215, G at position 236, V at position 237, L at position 238(if not T or R), T at position 240, I or A or V at position 245, Y atposition 249, V or I at position 252 (if not P), and V at position 254;or (2) Q at position 38, I at position 52, L at position 70, A atposition 82, P at position 83, V at position 84, W at position 86, R atposition 87, D at position 99, T at position 100, A at position 174, Sat position 189, S at position 195, R at position 203, and T at position222; or (3) H at position 37, I at position 75, K at position 107, A atposition 112, A at position 134, A at position 154, V at position 178(if not M or C), G at position 181, Y at position 186, T at position222, and V at position 245. The parentheticals in the above paragraphs(“if not X”) refers to the proviso in which the specified residue isexcluded from the specified amino acid residue position if amino acidresidue X is selected from paragraphs 076-077 for that residue position.

The HHDH polypeptides described in paragraphs 076-080 may have anycombination of two or more, three or more, four or more, five or more,six or more, seven or more, eight or more, nine or more, ten or more,eleven or more, twelve or more, thirteen or more, fourteen or more,fifteen or more, sixteen or more, seventeen more, eighteen or more,nineteen or more, or twenty or more of the above-described amino acidresidues (at the specified positions) in their sequences.

In a further embodiment, HHDH polypeptides of the present invention havethe sequence of SEQ ID NO: 2, with one or more substitutions selectedfrom the group consisting of I5V, T27A, A45V, E46Q, M54I, A60V, A65V,T67N, Q72H, V77I, Q87S, K91R, A93T, E95A, D96E, A100M/Q, A103T, S117R/T,Q118S, K121R/I/L/Y, P135S, W139R+N176E or W139D+N176R, T146A, C153N,Y166H, Y177G/A, L178M/C, H179N/D, S180D/T, E181K, D182N, E190V, V199A,H201W/Y, V205Y, V236L, W238T/R, L239F, I246V, G251E/A/S, and M252P.

In some embodiments, these HHDH polypeptides are encoded by an aminoacid sequence that comprises one or more substitutions selected from thesubgroup consisting of T27A, E46Q, M54I, A60V, A65V, Q87S, K91R, E95A,D96E, A100M/Q, S117R, Q118S, K121R, P135S, T146A, C153N, L178C, H179N,D182N, E190V, V199A, H201Y, V205Y, V236L, W238T, and G251E/A

In certain embodiments, these HHDH polypeptides are encoded by an aminoacid sequence having one or more substitutions selected from thesubgroup consisting of I5V, A45V, T67N, Q72H, V77I, A93T, A103T, S117T,K121I/Y, W139R+N176E or W139D+N176R, Y166H, Y177G/A, L178C, H179D,H201W, W238R, L239F, I246V, G251S, and M252P. In other embodiments,these HHDH polypeptides have an amino acid sequence that comprises oneor more substitutions selected from the group consisting of M54I, A60V,T67N, Q72H, A93T, A100M, S117T, K121R, P135S, T146A, Y177A/G, L178M/C,H179D, H201W, V205Y, W238R, L239F, and I246V. Some of these HHDHpolypeptides have an amino acid sequence that comprises one or moresubstitutions selected from the subgroup group consisting of T67N, Q72H,S117T, K121R, P135S, T146A, Y177A, H179D, H201W, V205Y, and I246V. HHDHpolypeptides of the present invention include those having an amino acidsequence comprising the following combination of amino acidsubstitutions: T67N+Q72H+S117T+K121R+P135S+T146A+T154A+Y177A+H179D+H201W+V205Y+I246V. In otherembodiments, the HHDH polypeptides have an amino acid sequence thatcomprises one or more amino acid substitutions selected from thesubgroup consisting of M541, A60V, K91E, A93T, A100M, Y177G, L178M/C,W238R, and L239F.

In addition to the amino acid substitutions described above, the HHDHpolypeptides described in paragraphs 082-084 may further comprise one ormore substitutions selected from the group consisting of S2T, T3A/P/S,A4V, V6D, V9I/F, K10L, G13S, G14S, M15K, G16C, S17T/R, R20C/S/K, A24T,H26Q, V28F, A29T, C30A, H31I, E33G, S34R, F35L, K36N, Q37H, K38Q, E40D,F44L, A45P (if not A45V from paragraphs 082-084), T47P/A, L51N, K52I,M54V (if not M54I), S55R, E56D, E58K, E61G/D, I63V, Y70L, Q72R (if notQ72H), V75I, L76P, S78C, D80Q, F82A/Y, A83P, P84S/L/V, E85A, F86W, Q87R,K91E (if not K91R), A93D (if not A93T), E95Q/G (if not E95A), D96N (ifnot D96E), G99D, A100T, R107K, V112A, A114T/S/G, V115A, S117P (if notS117R or T), K120N, K121E (if not K121R or I or L or Y), R122P, H126R,I130V, A133S, T134A/V, F136L/W/V, W139H (if not W139R or D), L142I/R,T144S, T146S (if not T146A), A152T, T154S/A, I168V, P169T, G174A, Y177F(if not Y177G or A), L178V (if not L178M or C), S180I, E181G/I, P184K,F186Y, T189S, T194L, N195S, H198N, V199M (if not V199A), K203R, K215E,A222T, V236G (if not V236L), F237V, W238L (if not W238T or R), A240T,M245I/A/V, W249Y, M252V/I (if not M252P), and E254V, wherein amino acidresidue position is determined by optimal alignment of the amino acidsequence of the HHDH polypeptide with SEQ ID NO: 2. Some of these HHDHpolypeptides have an amino acid sequence that further comprises one ormore substitutions selected from the subgroup consisting of Q37H, K38Q,K52I, Y70L, V75I, F82A, A83P, P84V, F86W, Q87R, G99D, A100T, R107K,V112A, T134A, T154A, G174A, L178V (if not L178M or C), E181G, F186Y,T189S, N195S, K203R, A222T, and M245V.

Some of the HHDH polypeptides of paragraph 082-084 further comprise oneor more substitutions selected from one of the following subgroups: (1)S2T, T3A/P/S, A4V, V6D, V9I/F, K10L, G13S, G14S, M15K, G16C, S17T/R,R20C/S/K, A24T, H26Q, V28F, A29T, C30A, H31I, E33G, S34R, F35L, K36N,Q37H, E40D, F44L, A45P (if not A45I from paragraphs 082-084), T47P/A,L51N, M54V (if not M54I), S55R, E56D, E58K, E61G/D, I63V, Q72R (if notQ72H), V75I, L76P, S78C, D80Q, F82Y, P84S/L, E85A, K91E (if not K91R),A93D (if not A93T), E95Q/G (if not E95A), D96N (if not D96E), R107K,V112A, A114T/S/G, V115A, S117P (if not S117R or T), K120N, K121E (if notK121R or I or L or Y), R122P, H126R, I130V, A133S, T134A/V, F136L/W/V,W139H (if not W139R or D), L142I/R, T144S, T146S (if not T146A), A152T,T154S/A, I168V, P169T, Y177F (if not Y177G or A), L178V (if not L178M orC), S180I, E181G/I, P184K, F186Y, T194L, H198N, V199M (if not V199A),K215E, V236G, F237V, W238L (if not W238T or R), A240T, M245I/A/V, W249Y,M252V/I (if not M252P), and E254V; or (2) K38Q, K52I, Y70L, F82A, A83P,P84V, F86W, Q87R, G99D, A100T, G174A, T189S, N195S, K203R, and A222T; or(3) Q37H, V75I, R107K, V112A, T134A, T154A, L178V (if not L178M or C),E181G, F186Y, A222T, and M245V. The parentheticals in the aboveparagraphs (“if not X”) refers to the proviso in which the specifiedsubstitution is excluded if the substitution X is selected fromparagraphs 082-084 for that specified residue position.

The present invention provides an HHDH polypeptide that is at least 80%identical to SEQ ID NO: 2, when optimally aligned with SEQ ID NO: 2, andwhich further has one or more substitutions selected from the groupconsisting of S2T, either T3A or T3P, A4V, V6D, either V9I or V9F, K10L,G13S, G14S, M15K, G16C, either S17T or S17R, either R20S, R20C or R20K,A24T, H26Q, V28F, A29T, C30A, H31L, E33G, S34R, F35L, K36N, Q37H, E40D,F44L, A45P, either T47P or T47A, K52N, M54V, S55R, E56D, E58K, eitherE61G or E61D, 163V, Q72R, V75I, L76P, S78C, F82Y, either P84S or P84L,E85Q, K91E, A93D, E95Q or E95G, D96N, R107K, V112A, either A114T orA114G or A114S, V115A, S117P, K120N, K121E, R122P, H126R, I130V, A133S,T134A or T134V, F136L or F136W or F136V, W139H, L142I or L142R, T144S,T146S, A152T, C153S, either T154S or T154A, I168V, P169T, Y177F, L178V,S180I, E181G or E181I, P184K, F186Y, T194I, H198N, V199M, K215E, V236G,F237V, W238L, A240T, either M254I or M245A or M245V, W249Y, M252V orM252I, and E254V. In some embodiments, HHDH polypeptides of the presentinvention are at least 85% identical to SEQ ID NO: 2 and have one ormore of the substitutions indicated above. Some HHDH polypeptides of thepresent invention are at least 90% identical to SEQ ID NO: 2, some areat least about 95% identical to SEQ ID NO: 2, and others are at least99% identical to sEQ ID NO: 2, all having one or more of thesubstitutions indicated above. Some of these HHDH polypeptides have atleast 2 or more of the aforementioned substitutions, and some of theseHHDH polypeptides have at least 3 or more of the aforementionedsubstitutions.

When optimally aligned with SEQ ID NO: 2, certain HHDH polypeptides ofthe present invention have a sequence corresponding to SEQ ID NO: 2,with one or more substitutions selected from the group consisting ofS2T, either T3A or T3P, A4V, V6D, either V9I or V9F, K10L, G13S, G14S,M15K, G16C, either S17T or S17R, either R20S, R20C or R20K, A24T, H26Q,V28F, A29T, C30A, H31L, E33G, S34R, F35L, K36N, Q37H, E40D, F44L, A45P,either T47P or T47A, K52N, M54V, S55R, E56D, E58K, either E61G or E61D,I63V, Q72R, V75I, L76P, S78C, F82Y, either P84S or P84L, E85Q, K91E,A93D, E95Q or E95G, D96N, V101I, R107K, V112A, either A114T or A114G orA114S, V115A, S117P, K120N, K121E, R122P, H126R, I130V, A133S, T134A orT134V, F136L or F136W or F136V, W139H, L142I or L142R, T144S, T146S,A152T, either T154S or T154A, I168V, P169T, Y177F or Y177A, L178V,S180I, E181G or E181I, P184K, F186Y, T194I, H198N, V199M, K215E, V236G,F237V, W238L, A240T, either M245I or M245A or M245V, W249Y, M252V orM252I, and E254V.

The HHDH polypeptides described in paragraph 088 may have anycombination of two or more, three or more, four or more, five or more,six or more, seven or more, eight or more, nine or more, ten or more,eleven or more, twelve or more, thirteen or more, fourteen or more,fifteen or more, sixteen or more, seventeen more, eighteen or more,nineteen or more, or twenty or more of the above-described amino acidsubstitutions in their sequences.

Exemplary HHDH polypeptides of the present invention are encoded by anucleic acid that hybridizes under stringent conditions oversubstantially the entire length of a nucleic acid corresponding to SEQID NO: 1, or complement thereof, wherein the encoded polypeptide has anamino acid sequence comprising one or more amino acid residues selectedfrom the group consisting of V at position 5, A at position 27, V atposition 45, Q at position 46, I at position 54, V at position 60, V atposition 65, N at position 67, H at position 72, I at position 77, S atposition 87, R at position 91, T at position 93, A at position 95, E atposition 96, M or Q at position 100, T at position 103, R or T atposition 117, S at position 118, R or I or L or Y at position 121, S atposition 135, R at position 139+E at position 176 or D at position 139+Rat position 176, A at position 146, N at position 153, H at position166, G or A at position 177, M or C at position 178, N or D at position179, D or T at position 180, K at position 181, N at position 182, V atposition 190, A at position 199, W or Y at position 201, Y at position205, L at position 236, T or R at position 238, F at position 239, V atposition 246, E or A or S at position 251, and P at position 252,wherein amino acid residue position is determined by optimal alignmentof the amino acid sequence with SEQ ID NO: 2 (see description inparagraphs 040-044).

The present invention also provides HHDH polypeptides encoded by anucleic acid that hybridizes under stringent conditions oversubstantially the entire length of a nucleic acid corresponding to SEQID NO: 1, where the encoded polypeptide, when optimally aligned with SEQID NO: 2, comprises an amino acid sequence having at least one aminoacid residue selected from the group consisting of T at (residue)position 2, A or P or S at position 3, V at position 4, D at position 6,either I or F at position 9, L at position 10, S at position 13, S atposition 14, K at position 15, C at position 16, T or R at position 17,either S or K at position 20, T at position 24, Q at position 26, F atposition 28, T at position 29, A at position 30, L at position 31, G atposition 33, R at position 34, L at position 35, N at position 36, H atposition 37, D at position 40, L at position 44, P at position 45,either P or A at position 47, N at position 52, V at position 54, R atposition 55, D at position 56, G or D at position 61, V at position 63,R or Q at position 72, I at position 75, P at position 76, C at position78, Y at position 82, either S or L at position 84, A at position 85, Eat position 91, D at position 93, Q or G at position 95, N at position96, I at position 101, K at position 107, A at position 112, either T, Sor G at position 114, A at position 115, P at position 117, N atposition 120, E at position 121, P at position 122, R at position 126, Vat position 130, S at position 133, A or V at position 134, L, W or V atposition 136, H at position 139, I or R at position 142, S at position144, S at position 146, T at position 152, either S or A at position154, V at position 168, T at position 169, F at position 177, V atposition 178, I at position 180, G or I at position 181, K at position184, Y at position 186, L at position 194, N at position 198, M atposition 199, E at position 215, G at position 236, V at position 237, Lat position 238, T at position 240, either I or A or V at position 245,Y at position 249, V or I at position 252, and V at position 254.

The HHDH polypeptides described in paragraphs 090-091 may have anycombination of two or more, three or more, four or more, five or more,six or more, seven or more, eight or more, nine or more, ten or more,eleven or more, twelve or more, thirteen or more, fourteen or more,fifteen or more, sixteen or more, seventeen more, eighteen or more,nineteen or more, or twenty or more of the above-described amino acidsubstitutions in their sequences.

Exemplary HHDH polypeptides of the present invention include an isolatedor recombinant polypeptide having HHDH activity, wherein the polypeptideis selected from the group consisting of a polypeptide comprising: (a)an amino acid sequence that is at least 80% identical (sometimes atleast 81% identical, 82% identical, 83% identical, 84% identical, 85%identical, 86% identical, 87% identical, 88% identical, 89% identical,90% identical, 91% identical, 92% identical, 93% identical, 94%identical, 95% identical, 96% identical, 97% identical, 98% or 98%identical) to SEQ ID NO: 730, wherein the amino acid sequence has one ormore amino acid residues selected from the group consisting of V atposition 5, A at position 27, V at position 45, Q at position 46, I atposition 54, V at position 60, V at position 65, N at position 67, H atposition 72, I at position 77, S at position 87, R at position 91, T atposition 93, A at position 95, E at position 96, M or Q at position 100,T at position 103, R or T at position 117, S at position 118, R or I orL or Y at position 121, S at position 135, R at position 139+E atposition 176 or D at position 139+R at position 176, A at position 146,N at position 153, H at position 166, G or A at position 177, M or C atposition 178, N or D at position 179, D or T at position 180, K atposition 181, N at position 182, V at position 190, A at position 199, Wor Y at position 201, Y at position 205, L at position 236, T or R atposition 238, F at position 239, V at position 246, E or A or S atposition 251, and P at position 252; and (b) an amino acid sequenceencoded by a nucleic acid that hybridizes under stringent conditionsover substantially the entire length of a nucleic acid corresponding toSEQ ID NO: 729, wherein the encoded polypeptide comprises an amino acidsequence having at least one amino acid residue selected from the groupconsisting of V at position 5, A at position 27, V at position 45, Q atposition 46, I at position 54, V at position 60, V at position 65, N atposition 67, H at position 72, I at position 77, S at position 87, R atposition 91, T at position 93, A at position 95, E at position 96, M orQ at position 100, T at position 103, R or T at position 117, S atposition 118, R or I or L or Y at position 121, S at position 135, R atposition 139+E at position 176 or D at position 139+R at position 176, Aat position 146, N at position 153, H at position 166, G or A atposition 177, M or C at position 178, N or D at position 179, D or T atposition 180, K at position 181, N at position 182, V at position 190, Aat position 199, W or Y at position 201, Y at position 205, L atposition 236, T or R at position 238, F at position 239, V at position246, E or A or S at position 251, and P at position 252, wherein for (a)and (b), amino acid residue position is determined by optimal alignmentof the amino acid sequence of the HHDH polypeptide with SEQ ID NO: 730.(see description in paragraphs 040-044)

In another embodiment, the present invention is directed to an isolatedor recombinant polypeptide having HHDH activity, wherein the polypeptidecomprises an amino acid sequence that is selected from the groupconsisting of: (a) an amino acid sequence that is at least 80% identicalto SEQ ID NO: 730 (typically at least 85% identical, more typically atleast 90% identical, preferably at least 95% identical, more preferablyat least 97% identical, even more preferably at least 98% identical, andmost preferably at least 99% identical to SEQ ID NO: 730) and has one ormore amino acid residues selected from the group consisting of Q atposition 38, I at position 54, M at position 100, R at position 121, Sat position 135, A at position 146, T at position 238, and E at position251; (b) an amino acid sequence encoded by a nucleic acid thathybridizes under stringent conditions over substantially the entirelength of a nucleic acid corresponding to SEQ ID NO: 729, and whereinthe encoded polypeptide, when optimally aligned with SEQ ID NO: 730,comprises an amino acid sequence having at least one amino acid residueselected from the group consisting of I at position 54, M at position100, R at position 121, S at position 135, A at position 146, T atposition 238, and E at position 251.

In another embodiment, the polypeptide of the preceding paragraph hasone or more amino acid residues selected from the group consisting of Iat position 54, M at position 100, R at position 121, S at position 135,A at position 146, T at position 238, and E at position 251.

In a specific embodiment, the HHDH polypeptides of paragraph 094 have anamino acid sequence comprising one or more amino acid residues selectedfrom the group consisting of Q at position 38, I at position 54, M atposition 100, R at position 121, S at position 135, A at position 146, Tat position 238, and E at position 251.

In another embodiment, the present invention is directed to thepolypeptide of SEQ ID NO: 730 having an amino acid sequence with one ormore amino acid residue substitutions selected from the group consistingof M541, T 100M, K121R, P135S, S146A, W238T, and G251E.

The HHDH polypeptides of paragraphs 094-097 may have any combination oftwo or more, three or more, four or more, five or more, six or more, orseven or eight of the above-described substitutions or amino acidresidues (at the specified positions) in their sequences.

In another aspect, the present invention is directed to an isolated orrecombinant polypeptide having HHDH activity, wherein the polypeptidecomprises an amino acid sequence that is at least 86% identical(sometimes at least 87% identical, 88% identical, 89% identical, 90%identical, 91% identical, 92% identical, 93% identical, 94% identical,95% identical, 96% identical, 97% identical, 98% identical, and at least99% identical) to a reference sequence selected from the groupconsisting of SEQ ID NO: 750 or SEQ ID NO: 2848.

HHDH polypeptides described in paragraph 099 may have an amino acidsequence that comprises one or more amino acid residues selected fromthe group consisting of A T at position 2, A or P or S at position 3, Vat position 4, D at position 6, I or F at position 9, L at position 10,S at position 13, S at position 14, K at position 15, C at position 16,T or R at position 17, C or S or K at position 20, T at position 24, Qat position 26, F at position 28, T at position 29, A at position 30, Iat position 31, G at position 33, R at position 34, L at position 35, Nat position 36, H at position 37, D at position 40, L at position 44, Pat position 45, P or A at position 47, N at position 51, V at position54, R at position 55, D at position 56, K at position 58, G or D atposition 61, V at position 63, R at position 72, I at position 75, P atposition 76, C at position 78, Q at position 80, Y at position 82, S orL at position 84, A at position 85, E at position 91, D at position 93,Q or G at position 95, N at position 96, K at position 107, A atposition 112, T or S or G at position 114, A at position 115, P atposition 117, N at position 120, E at position 121, P at position 122, Rat position 126, V at position 130, S at position 133, A or V atposition 134, L or W or V at position 136, H at position 139, I or R atposition 142, S at position 144, S at position 146, T at position 152, Sor A at position 154, V at position 168, T at position 169, F atposition 177, V at position 178, I at position 180, G or I at position181, K at position 184, Y at position 186, L at position 194, N atposition 198, M at position 199, E at position 215, G at position 236, Vat position 237, L at position 238, T at position 240, I or A or V atposition 245, Y at position 249, V or I at position 252, and V atposition 254.

Typically, the HHDH polypeptides of paragraph 099 have an amino acidsequence that comprises one or more amino acid residues selected fromthe group consisting of V at position 5, A at position 27, V at position45, Q at position 46, I at position 54, V at position 60, V at position65, N at position 67, H at position 72, I at position 77, S at position87, R at position 91, T at position 93, A at position 95, E at position96, M or Q at position 100, T at position 103, R or T at position 117, Sat position 118, R or I or L or Y at position 121, S at position 135, Rat position 139+E at position 176 or D at position 139+R at position176, A at position 146, N at position 153, H at position 166, G or A atposition 177, M or C at position 178, N or D at position 179, D or T atposition 180, K at position 181, N at position 182, V at position 190, Aat position 199, W or Y at position 201, Y at position 205, L atposition 236, T or R at position 238, F at position 239, V at position246, E or A or S at position 251, and P at position 252, wherein aminoacid residue position is determined by optimal alignment of the aminoacid sequence of the HHDH polypeptide with the reference sequence. Insome embodiments, the above-described HHDH polypeptides are encoded byan amino acid sequence that comprises one or more amino acid residuesselected from the subgroup consisting of A at position 27, Q at position46, I at position 54, V at position 60, V at position 65, S at position87, R at position 91, A at position 95, E at position 96, M or Q atposition 100, R at position 117, S at position 118, R at position 121, Sat position 135, A at position 146, N at position 153, M at position178, N at position 179, N at position 182, V at position 190, A atposition 199, Y at position 201, Y at position 205, L at position 236, Tat position 238, and E or A at position 251.

In certain embodiments, these HHDH polypeptides are encoded by an aminoacid sequence having one or more amino acid residues selected from thesubgroup consisting of V at position 5, V at position 45, N at position67, H at position 72, I at position 77, T at position 93, T at position103, T at position 117, S at position 118, I or L or Y at position 121,Rat position 139+E at position 176 or D at position 139+R at position176, H at position 166, G or A at position 177, C at position 178, D atposition 179, W at position 201, R at position 238, F at position 239, Vat position 246, S at position 251, and P at position 252. In otherembodiments, these HHDH polypeptides are encoded by an amino acidsequence that comprises one or more residues selected from the groupconsisting of I at position 54, V at position 60, N at position 67, H atposition 72, T at position 93, M at position 100, T at position 117, Rat position 121, S at position 135, A at position 146, A or G atposition 177, M or C at position 178, D at position 179, W at position201, Y at position 205, R at position 238, F at position 239, and V atposition 246. Some of these HHDH polypeptides have an amino acidsequence that comprises one or more amino acid residues selected fromthe subgroup consisting of N at position 67, H at position 72, T atposition 117, R at position 121, S at position 135, A at position 146, Aat position 177, D at position 179, W at position 201, Y at position205, and V at position 246. HHDH polypeptides of the present inventioninclude those having an amino acid sequence comprising the followingcombination of amino acid residues: N at position 67+H at position 72+Tat position 117+R at position 121+S at position 135+A at position 146+Aat position 154+A at position 177+D at position 179+W at position 201+Yat position 205+V at position 246. In other embodiments the HHDHpolypeptides have an amino acid sequence that comprises one or moreamino acid residues selected from the group consisting of I at position54, V at position 60, E at position 91, T at position 93, M at position100, G at position 177, M or C at position 178, R at position 238, and Fat position 239.

In addition to the amino acid residues described above, the HHDHpolypeptides described in paragraphs 0101-0102 may further comprise oneor more amino acid residues selected from the group consisting of T atposition 2, A or P or S at position 3, V at position 4, D at position 6,I or F at position 9, L at position 10, S at position 13, S at position14, K at position 15, C at position 16, T or R at position 17, C or S orK at position 20, T at position 24, Q at position 26, F at position 28,T at position 29, A at position 30, I at position 31, G at position 33,R at position 34, L at position 35, N at position 36, H at position 37,Q at position 38, D at position 40, L at position 44, P at position 45(if not V from paragraphs 0101-0102), P or A at position 47, N atposition 51, I at position 52, V at position 54 (if not I), R atposition 55, D at position 56, K at position 58, G or D at position 61,V at position 63, L at position 70, R at position 72 (if not H), I atposition 75, P at position 76, C at position 78, Q at position 80, A orY at position 82, P at position 83, S or L or V at position 84, A atposition 85, W at position 86, R at position 87, E at position 91 (ifnot R), D at position 93 (if not T), Q or G at position 95 (if not A), Nat position 96 (if not E), D at position 99, T at position 100, K atposition 107, A at position 112, T or S or G at position 114, A atposition 115, P at position 117 (if not R or T), N at position 120, E atposition 121 (if not R or I or L or Y), P at position 122, R at position126, V at position 130, S at position 133, A or V at position 134, L orW or V at position 136, H at position 139 (if not R or D), I or R atposition 142, S at position 144, S at position 146 (if not A), T atposition 152, S or A at position 154, V at position 168, T at position169, A at position 174, F at position 177 (if not G or A), V at position178 (if not M or C), I at position 180, G or I at position 181, K atposition 184, Y at position 186, S at position 189, L at position 194, Sat position 195, N at position 198, M at position 199 (if not A), R atposition 203, E at position 215, T at position 222, G at position 236(if not L), V at position 237, L at position 238 (if not T or R), T atposition 240, I or A or V at position 245, Y at position 249, V or I atposition 252 (if not P), and V at position 254, wherein amino acidresidue position is determined by optimal alignment of the amino acidsequence of the HHDH polypeptide with the reference sequence. Some ofthese HHDH polypeptides have an amino acid sequence that furthercomprises one or more amino acid residues selected from the subgroupconsisting of H at position 37, Q at position 38, I at position 52, L atposition 70, I at position 75, A at position 82, P at position 83, V atposition 84, W at position 86, R at position 87, D at position 99, T atposition 100, K at position 107, A at position 112, A at position 134, Aat position 154, A at position 174, V at position 178 (if not M or C), Gat position 181, Y at position 186, S at position 189, S at position195, R at position 203, T at position 222, and V at position 245.

Some of the HHDH polypeptides of paragraphs 0101-0102 may furthercomprise one or more amino acid residues selected from one of thefollowing subgroups: (1) T at position 2, A or P or S at position 3, Vat position 4, D at position 6, I or F at position 9, L at position 10,S at position 13, S at position 14, K at position 15, C at position 16,T or R at position 17, C or S or K at position 20, T at position 24, Qat position 26, F at position 28, T at position 29, A at position 30, Iat position 31, G at position 33, R at position 34, L at position 35, Nat position 36, H at position 37, D at position 40, L at position 44, Pat position 45 (if not I), P or A at position 47, N at position 51, V atposition 54 (if not I), R at position 55, D at position 56, K atposition 58, G or D at position 61, V at position 63, R at position 72(if not H), I at position 75, P at position 76, C at position 78, Q atposition 80, Y at position 82, S or L at position 84, A at position 85,E at position 91 (if not R), D at position 93 (if not T), Q or G atposition 95 (if not A), N at position 96 (if not E), K at position 107,A at position 112, T or S or G at position 114, A at position 115, P atposition 117 (if not R or T), N at position 120, E at position 121 (ifnot R or I or L or Y), P at position 122, R at position 126, V atposition 130, S at position 133, A or V at position 134, L or W or V atposition 136, H at position 139 (if not R or D), I or R at position 142,S at position 144, S at position 146 (if not A), T at position 152, S orA at position 154, V at position 168, T at position 169, F at position177 (if not G or A), V at position 178 (if not M or C), I at position180, G or I at position 181, K at position 184, Y at position 186, L atposition 194, N at position 198, M at position 199 (if not A), E atposition 215, G at position 236, V at position 237, L at position 238(if not T or R), T at position 240, I or A or V at position 245, Y atposition 249, V or I at position 252 (if not P), and V at position 254;or (2) Q at position 38, I at position 52, L at position 70, A atposition 82, P at position 83, V at position 84, W at position 86, R atposition 87, D at position 99, T at position 100, A at position 174, Sat position 189, S at position 195, R at position 203, and T at position222; or (3) H at position 37, I at position 75, K at position 107, A atposition 112, A at position 134, A at position 154, V at position 178(if not M or C), G at position 181, Y at position 186, T at position222, and V at position 245. The parentheticals in the above paragraphs(“if not X”) refers to the proviso in which the specified residue isexcluded from the specified amino acid residue position if amino acidresidue X is selected from paragraphs 0101-0102 for that residueposition.

Exemplary HHDH polypeptides of the present invention include an isolatedor recombinant HHDH polypeptide having HHDH activity, wherein thepolypeptide is selected from the group consisting of a polypeptidecomprising: (a) an amino acid sequence that is at least 80% identical toa reference sequence selected from the group consisting of SEQ ID NO:750 and SEQ ID NO: 2848 (typically at least 81% identical, 82%identical, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, 98%, or at least 99% identical) to thereference sequence and that has one or more amino acid residues selectedfrom V at position 5, A at position 27, V at position 45, Q at position46, I at position 54, V at position 60, V at position 65, N at position67, H at position 72, I at position 77, S at position 87, R at position91, T at position 93, A at position 95, E at position 96, M or Q atposition 100, T at position 103, R or T at position 117, S at position118, R or I or L or Y at position 121, S at position 135, R at position139+E at position 176 or D at position 139+R at position 176, A atposition 146, N at position 153, H at position 166, G or A at position177, M or C at position 178, N or D at position 179, D or T at position180, K at position 181, N at position 182, V at position 190, A atposition 199, W or Y at position 201, Y at position 205, L at position236, T or R at position 238, F at position 239, V at position 246, E orA or S at position 251, and P at position 252; and (b) an amino acidsequence encoded by a nucleic acid that hybridizes under stringentconditions over substantially the entire length of a nucleic acidcorresponding to a reference nucleic acid sequence selected from thegroup consisting of SEQ ID NO: 749, SEQ ID NO: 2847, and complementarysequences thereof, wherein the encoded polypeptide comprises an aminoacid sequence having at least one amino acid residue selected from thegroup consisting of V at position 5, A at position 27, V at position 45,Q at position 46, I at position 54, V at position 60, V at position 65,N at position 67, H at position 72, I at position 77, S at position 87,R at position 91, T at position 93, A at position 95, E at position 96,M or Q at position 100, T at position 103, R or T at position 117, S atposition 118, R or I or L or Y at position 121, S at position 135, R atposition 139+E at position 176 or D at position 139+R at position 176, Aat position 146, N at position 153, H at position 166, G or A atposition 177, M or C at position 178, N or D at position 179, D or T atposition 180, K at position 181, N at position 182, V at position 190, Aat position 199, W or Y at position 201, Y at position 205, L atposition 236, T or R at position 238, F at position 239, V at position246, E or A or S at position 251, and P at position 252, wherein aminoacid residue position is determined by optimal alignment of the aminoacid sequence of the HHDH polypeptide with a reference amino acidsequence selected from the group consisting of SEQ ID NO: 750 and SEQ IDNO: 2848 (i.e., corresponding to that encoded by the reference nucleicacid sequence). (See description in paragraphs 040-044)

In a specific embodiment, the present invention is directed to anisolated or recombinant polypeptide having HHDH activity, wherein thepolypeptide comprises an amino acid sequence selected from the groupconsisting of: (a) an amino acid sequence that is at least 80% identicalto a reference sequence selected from the group consisting of SEQ ID NO:750 and SEQ ID NO: 2848 (typically at least 85% identical, moretypically at least 90% identical, preferably at least 95% identical,more preferably at least 97% identical, even more preferably at least98% or 99% identical to SEQ ID NO: 750 or SEQ ID NO: 2848) and that hasone or more amino acid residues selected from the group consisting of Aat position 27, Q at position 46, I at position 54, V at position 60, Vat position 65, S at position 87, R at position 91, A at position 95, Eat position 96, M or Q at position 100, R at position 117, S at position118, E at position 121, S at position 135, S at position 144, A atposition 146, T at position 152, N at position 153, M at position 178, Nat position 179, N at position 182, V at position 190, A at position199, Y at position 201, Y at position 205, L at position 236, T atposition 238, and E or A at position 251; and (b) an amino acid sequenceencoded by a nucleic acid that hybridizes under stringent conditionsover substantially the entire length of a reference nucleic acidcorresponding to SEQ ID NO: 749 or SEQ ID NO: 2847, and wherein theencoded polypeptide, when optimally aligned with a reference polypeptidecorresponding to SEQ ID NO: 750 or SEQ ID NO: 2848, respectively,comprises an amino acid sequence having at least one amino acid residueselected from the group consisting of A at position 27, Q at position46, I at position 54, V at position 60, V at position 65, S at position87, R at position 91, A at position 95, E at position 96, M or Q atposition 100, R at position 117, S at position 118, E at position 121, Sat position 135, S at position 144, A at position 146, T at position152, N at position 153, M at position 178, N at position 179, N atposition 182, V at position 190, A at position 199, Y at position 201, Yat position 205, L at position 236, T at position 238, and E or A atposition 251.

In yet another embodiment, the present invention is directed to apolypeptide having HHDH enzymatic activity and having an amino acidsequence is at least 80% identical to a reference sequence selected fromthe group consisting of SEQ ID NO: 750 and SEQ ID NO: 2848 (typically atleast 85% identical, more typically at least 90% identical, preferablyat least 95% identical, more preferably at least 97% identical, evenmore preferably at least 98% identical, and most preferably at least 99%identical to SEQ ID NO: 750 or SEQ ID NO: 2848) and comprises two ormore residues selected from the group consisting of A at position 27, Qat position 46, I at position 54, V at position 60, S at position 87, Rat position 91, A at position 95, E at position 96, M or Q at position100, R at position 117, S at position 118, R at position 121, S atposition 135, S at position 144, A at position 146, N at position 153, Mat position 178, N at position 179, N at position 182, V at position190, A at position 199, Y at position 201, L at position 236, T atposition 238, and E or A at position 251.

In another embodiment, the present invention is directed to an isolatedor recombinant polypeptide having HHDH activity, wherein the polypeptidecomprises an amino acid sequence selected from the group consisting of:(a) an amino acid sequence that is at least 80% identical to a referencesequence selected from the group consisting of SEQ ID NO: 750 and SEQ IDNO: 2848 (typically at least 85% identical, more typically at least 90%identical, preferably at least 95% identical, more preferably at least97% identical, even more preferably at least 98% identical, and mostpreferably at least 99% identical to SEQ ID NO: 750 or SEQ ID NO: 2848)and that has two or more residues are selected from the group consistingof A at position 27, Q at position 38, Q at position 46, I at position54, V at position 60, S at position 87, R at position 91, A at position95, E at position 96, M or Q at position 100, R at position 117, S atposition 118, R at position 121, S at position 135, S at position 144, Aat position 146, N at position 153, M at position 178, N at position179, N at position 182, V at position 190, A at position 199, Y atposition 201, L at position 236, T at position 238, and E or A atposition 251; and (b) an amino acid sequence encoded by a nucleic acidthat hybridizes under stringent conditions over substantially the entirelength of a reference nucleic acid corresponding to SEQ ID NO: 749 orSEQ ID NO: 2847, and wherein the encoded polypeptide, when optimallyaligned with SEQ ID NO: 750 or SEQ ID NO: 2848, respectively, comprisesan amino acid sequence having two or more residues are selected from thegroup consisting of A at position 27, Q at position 38, Q at position46, I at position 54, V at position 60, S at position 87, R at position91, A at position 95, E at position 96, M or Q at position 100, R atposition 117, S at position 118, R at position 121, S at position 135, Sat position 144, A at position 146, N at position 153, M at position178, N at position 179, N at position 182, V at position 190, A atposition 199, Y at position 201, L at position 236, T at position 238,and E or A at position 251. In a more preferred embodiment, the “two ormore residues” are three or more residues. In an even more preferredembodiment, the three or more residues are the following three residues:R at position 121, S at position 135, and A at position 146.

In another embodiment, the present invention is also directed to anisolated or recombinant polypeptide having HHDH activity, wherein thepolypeptide is at least 97% identical to SEQ ID NO: 750 or SEQ ID NO:2848, and wherein the amino acid sequence of the polypeptide comprisesone or more amino acid residue selected from the group consisting of Aat position 27, Q at position 38, Q at position 46, I at position 54, Vat position 60, S at position 87, R at position 91, A at position 95, Eat position 96, M or Q at position 100, R at position 117, S at position118, R at position 121, S at position 135, S at position 144, A atposition 146, Nat position 153, Mat position 178, N at position 179, Nat position 182, V at position 190, A at position 199, Y at position201, L at position 236, T at position 238, and E or A at position 251.

The HHDH polypeptides described in paragraphs 0105-0109 may have anycombination of two or more, three or more, four or more, five or more,six or more, seven or more, eight or more, nine or more, ten or more,eleven or more, twelve or more, thirteen or more, fourteen or more,fifteen or more, sixteen or more, seventeen more, eighteen or more,nineteen or more, or twenty or more of the above-described amino acidresidues (at the specified positions) in their sequences.

Exemplary HHDH polypeptides of the present invention include an isolatedor recombinant HHDH polypeptide having an amino acid sequence selectedfrom the group consisting of: (a) an amino acid sequence that is atleast 80% identical (sometimes at least 81% identical, 82% identical,83% identical, 84% identical, 85% identical, 86% identical, 87%identical, 88% identical, 89% identical, 90% identical, 91% identical,92% identical, 93% identical, 94% identical, 95% identical, 96%identical, 97% identical, 98% or 99% identical to a reference sequenceselected from the group consisting of SEQ ID NO: 2040, SEQ ID NO: 2130,SEQ ID NO: 1926, SEQ ID NO: 2348, and SEQ ID NO: 1984, wherein the aminoacid sequence has one or more amino acid residues selected from thegroup consisting of V at position 5, A at position 27, V at position 45,Q at position 46, I at position 54, V at position 60, V at position 65,N at position 67, H at position 72, I at position 77, S at position 87,R at position 91, T at position 93, A at position 95, E at position 96,M or Q at position 100, T at position 103, R or T at position 117, S atposition 118, R or I or L or Y at position 121, S at position 135, R atposition 139+E at position 176 or D at position 139+R at position 176, Aat position 146, N at position 153, H at position 166, G or A atposition 177, M or C at position 178, N or D at position 179, D or T atposition 180, K at position 181, N at position 182, V at position 190, Aat position 199, W or Y at position 201, Y at position 205, L atposition 236, T or R at position 238, F at position 239, V at position246, E or A or S at position 251, and P at position 25.2; and (b) anamino acid sequence encoded by a nucleic acid that hybridizes understringent conditions over substantially the entire length of a nucleicacid corresponding to a reference nucleic acid sequence selected fromthe group consisting of SEQ ID NO: 2039, SEQ ID NO: 2129, SEQ ID NO:1925, SEQ ID NO: 2347, SEQ ID NO: 1983, and complementary sequencesthereof, wherein the encoded polypeptide comprises an amino acidsequence having at one amino acid residue selected from the groupconsisting of V at position 5, A at position 27, V at position 45, Q atposition 46, I at position 54, V at position 60, V at position 65, N atposition 67, H at position 72, I at position 77, S at position 87, R atposition 91, T at position 93, A at position 95, E at position 96, M orQ at position 100, T at position 103, R or T at position 117, S atposition 118, R or I or L or Y at position 121, S at position 135, R atposition 139+E at position 176 or D at position 139+R at position 176, Aat position 146, N at position 153, H at position 166, G or A atposition 177, M or C at position 178, N or D at position 179, D or T atposition 180, K at position 181, N at position 182, V at position 190, Aat position 199, W or Y at position 201, Y at position 205, L atposition 236, T or R at position 238, F at position 239, V at position246, E or A or S at position 251, and P at position 252; and wherein in(a) and (b), amino acid residue position is determined by optimalalignment of the amino acid sequence of the HHDH polypeptide with areference amino acid sequence that is the amino acid sequence encoded bythe reference nucleic acid sequence (i.e., SEQ ID NO: 2040, 2130, 1926,2348, and 1984, respectively). (See description in paragraphs 040-044)

Significantly, HHDH polypeptides of the present invention include thosehaving improved (i.e., greater) HHDH enzymatic activity relative to thewild-type HHDH of SEQ ID NO: 2, as measured in the assay described inExample 5A. For example, HHDH polypeptides of the present inventionoften have HHDH activity that is at least 1.4 fold greater HHDH activityas compared to wild-type HHDH having the amino acid sequence of SEQ IDNO: 2, as measured in the assay described in Example 5A.

Some HHDH polypeptides of the present invention (including, for example,SEQ ID NOS: 740, 742, 728, 90, 92, 94, 96, and many others describedherein) have HHDH activity that is at least 2 fold and often at least2.4 fold, up to 100 fold greater than the HHDH activity of Agrobacteriumsp. HHDH (SEQ ID NO: 2); the HHDH polypeptides of the present inventionalso includes those having HHDH activity that is from 100 to 500 foldgreater than the activity of Agrobacterium sp. HHDH (including, forexample, SEQ ID NOS: 100, 732, 734, 736 and many others describedherein); and some HHDH polypeptides have HHDH enzyme activity that is500 to 1000 times greater than the activity of Agrobacterium sp. HHDH(including, for example, SEQ ID NOS: 726, 730, and many others describedherein), the enzyme activities being measured in the assay described inExample 5A. Therefore, the isolated or recombinant HHDH polypeptidesdescribed herein include those having at least 1.4 fold greater(typically 1.4 fold to 10,000 fold greater, more typically 1.4 fold to1000 fold greater) HHDH activity as compared to wild-type HHDH havingthe amino acid sequence of SEQ ID NO: 2, as measured in the assaydescribed in Example 5A.

In a specific embodiment, the present invention is directed to apolypeptide, typically an isolated or recombinant polypeptide havingHHDH activity greater than the wild-type HHDH of SEQ ID NO. 2, andhaving an amino acid sequence that is at least 89% identical to SEQ IDNO: 442; typically, 93% identical to SEQ ID NO: 442; more typically, 95%identical to SEQ ID NO: 442; even more typically, 97% identical to SEQID NO: 442; most typically, 99% identical to SEQ ID NO: 442.

In another embodiment, the present invention is directed to apolypeptide, typically an isolated or recombinant polypeptide havingHHDH activity greater than the wild-type HHDH of SEQ ID NO. 2, andhaving an amino acid sequence that is at least 88% identical to SEQ IDNO: 702; typically, 93% identical to SEQ ID NO: 702: more typically, 95%identical to SEQ ID NO: 702; even more typically, 97% identical to SEQID NO: 702; most typically, 99% identical to SEQ ID NO: 702.

In a further embodiment, the present invention provides an HHDHpolypeptide having an amino acid sequence that is at least 96% identicalto SEQ ID NO: 116 or 448. HHDH polypeptides of the present inventioninclude those that are least 97% identical, 98% identical, and 99%identical to SEQ ID NO: 116 or 448.

The present invention further provides an HHDH polypeptide having anamino acid sequence that is at least 95% identical to SEQ ID NO: 264,266, 470 or 476. Desirable HHDH polypeptides of the present inventioninclude those that are least 96% identical, 97% identical, 98%identical, and 99% identical to SEQ ID NO: 264, 266, 470 or 476.

The present invention includes HHDH polypeptides that catalyze theconversion of ECHB to HN under commercially relevant process conditions.Example 5D provides an assay for evaluating the ability of the HHDHpolypeptides to catalyze the conversion of ECHB to HN product undercommercially relevant process conditions. Many of the HHDH polypeptidesdescribed herein exhibit superior HHDH activity under commerciallyrelevant conditions as evaluated in the assay of Example 5D. Therefore,HHDH polypeptides of the present invention include those exhibitingdetectable HHDH activity in the assay of Example 5D. Many of the HHDHpolypeptides described herein exhibit superior activity to the improvedHHDH polypeptide corresponding to SEQ ID NO: 730, in the assay ofExample 5D, where the HHDH polypeptide of SEQ ID NO: 730 itself exhibitssuperior activity relative to wildtype HHDH (SEQ ID NO: 2) in the assayof Example 5A. Therefore, HHDH polypeptides of the present inventioninclude those having greater activity than the HHDH polypeptide of SEQID NO: 730 under the commercially relevant conditions of the assaydescribed in Example 5D. Many exhibit at least 1.5-fold greater activitythan the HHDH polypeptide of SEQ ID NO: 730 in the assay of Example 5D.

Some of the HHDH polypeptides of the previous paragraph have about2-fold greater HHDH activity compared to the HHDH polypeptide of SEQ IDNO: 730, some at least 2.5-fold, 3-fold, 4-fold, up to about 10-fold or100-fold HHDH activity over the HHDH polypeptide corresponding to SEQ IDNO: 730, as measured in the assay of Example 5D.

In a specific embodiment, the present invention provides an isolated orrecombinant HHDH polypeptide comprising an amino acid sequence that isat least 75% identical to a reference sequence selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO: 750, SEQ ID NO: 1926, SEQ ID NO:1984, SEQ ID NO: 2040, SEQ ID NO: 2130, SEQ ID NO: 2348, and SEQ ID NO:2848 (sometimes at least 76% identical, 77% identical, 78% identical,79% identical, 80% identical, 85% identical, 86% identical, 87%identical, 88% identical, 89% identical, 90% identical, 91% identical,92% identical, 93%, identical, 94% identical, 95% identical, 95%identical, 96% identical, 98% identical, or 99% identical to thereference sequence), wherein the HHDH polypeptide has at least about1.5-fold HHDH activity over the HHDH polypeptide corresponding to SEQ IDNO: 730, as measured in the assay of Example 5D.

In some embodiments, the HHDH polypeptides of paragraph 0120 have aminoacid sequences that comprise one or more amino acid residues selectedfrom the group consisting of Q at position 38, I at position 52, L atposition 70, A at position 82, P at position 83, V at position 84, W atposition 86, R at position 87, D at position 99, T at position 100, A atposition 174, S at position 189, S at position 195, R at position 203,and T at position 222, wherein amino acid position is determined byoptimal alignment of the amino acid sequence of the HHDH polypeptidewith SEQ ID NO: 2. These HHDH polypeptides may have any combination oftwo or more, three or more, four or more, five or more, six or more,seven or more, eight or more, nine or more, ten or more, eleven or more,twelve or more, thirteen or more, fourteen or more, or all fifteen ofthese amino acid residues (at the specified position) in theirsequences. In certain embodiments, these HHDH polypeptides comprise oneor more amino acid residues selected from the group consisting of Q atposition 38, I at position 52, L at position 70, A at position 82, P atposition 83, V at position 84, W at position 86, D at position 99, T atposition 100, A at position 174, S at position 189, S at position 195,and R at position 203. These HHDH polypeptides may have any combinationof two or more, three or more, four or more, five or more, six or more,seven or more, eight or more, nine or more, ten or more, eleven or more,twelve or more, or all thirteen of these amino acid residues (at thespecified position) in their sequences.

In another embodiment, HHDH polypeptides comprise the sequence of SEQ IDNO: 2, and one or more substitutions selected from each of the followingtwo substitution groups: (a) M54I, A60V, K91E, A93T, A100M, Y177G,L178M/C, W238R, and L239F; and (b) S2T, T3A/P/S, A4V, 15V, V6D, V9I/F,K10L, G13S, G14S, M15K, G16C, S17T/R, R20C/S/K, A24T, H26Q, T27A, V28F,A29T, C30A, H31I, E33G, S34R, F35L, K36N, Q37H, E40D, F44L, A45P/V,E46Q, T47P/A, L51N, M54V/I, S55R, E56D, E58K, A60V, E61G/D, I63V, A65V,T67N, Q72R/H, V75I, L76P, V77I, S78C, D80Q, F82Y, P84S/L, E85A, Q87S,K91E/R, A93D/T, E95Q/G/A, D96N/E, A100M/Q, A103T, R107K, V112A,A114T/S/G, V115A, S117P/R/T, Q118S, K120N, K121E/R/I/L/Y, R122P, H126R,I130V, A133S, T134A/V, P135S, F136L/W/V, W139H or W139R+N176E orW139D+N176R, L142I/R, T144S, T146S/A, A152T, C153N, T154S/A, Y166H,I168V, P169T, Y177F/G/A, L178V/M/C, H179N/D, S180I/D/T, E181G/I/K,D182N, P184K, F186Y, E190V, T194L, H198N, V199M/A, H201W/Y, V205Y,K215E, V236G/L, F237V, W238L/T/R, L239F, A240T, M245V/A/V, I246V, W249Y,G251E/A/S, M252V/I/P, and E254V, where the HHDH polypeptide has at leastabout 1.5-fold greater HHDH activity compared to the HHDH polypeptidecorresponding to SEQ ID NO: 730, as measured in the assay of Example 5D.

The amino acid sequences of the HHDH polypeptides described in the aboveparagraph may have: any combination of two or more, three or more, fouror more, five or more, six or more, seven or more, eight or more, nineor more, ten or more, eleven or more, twelve or more, thirteen or more,fourteen or more, or all fifteen of the substitutions of group (a); andany combination of two or more, three or more, four or more, five ormore, six or more, seven or more, eight or more, nine or more, ten ormore, eleven or more, twelve or more, thirteen or more, fourteen ormore, fifteen or more, sixteen or more, seventeen or more, eighteen ormore, nineteen or more, or twenty or more of the substitutions listed ingroup (b).

Some of the HHDH polypeptides of the paragraphs 0120-0123 have about2-fold greater HHDH activity compared to the HHDH polypeptide of SEQ IDNO: 730, some at least 2.5-fold, 3-fold, 4-fold, up to about 10-fold or100-fold greater HHDH activity than the HHDH polypeptide correspondingto SEQ ID NO: 730, as measured in the assay of Example 5D.

HHDH polypeptides of the present invention include those encoded by anucleic acid that hybridizes under stringent conditions oversubstantially the entire length of a nucleic acid corresponding to areference nucleic acid sequence selected from the group consisting of areference nucleic acid sequence selected from the group consisting of 1,3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 33, 35, 37, 39, 41,43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77,79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137,139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165,167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193,195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221,223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249,251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277,279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305,307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333,335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361,363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389,391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417,419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445,447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 4712, 473,475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501,503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529,531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557,559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585,587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613,615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641,643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669,671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697,699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725,727, 729, 731, 733, 735, 739, 741, 743, 745, 747, 749, 751, 753, 755,757, 759, 761, 763, 765, 767, 769, 771, 773, 775, 777, 779, 781, 783,785, 787, 789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 809, 811,813, 815, 817, 819, 821, 823, 825, 827, 829, 831, 833, 835, 837, 839,841, 843, 845, 847, 849, 851, 853, 855, 857, 859, 861, 863, 865, 867,869, 871, 873, 875, 877, 879, 881, 883, 885, 887, 889, 891, 893, 895,897, 899, 901, 903, 905, 907, 909, 911, 913, 915, 917, 919, 921, 923,925, 927, 929, 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951,953, 955, 957, 959, 961, 963, 965, 967, 969, 971, 973, 975, 977, 979,981, 983, 985, 987, 989, 991, 993, 995, 997, 999, 1001, 1103, 1005,1007, 1009, 1011, 1013, 1015, 1017, 1019, 1021, 1023, 1025, 1027, 1029,1031, 1033, 1035, 1037, 1039, 1041, 1043, 1045, 1047, 1049, 1051, 1053,1055, 1057, 1059, 1061, 1063, 1065, 1067, 1069, 1071, 1073, 1075, 1077,1079, 1081, 1083, 1085, 1087, 1089, 1091, 1093, 1095, 1097, 1099, 1101,1103, 1105, 1107, 1109, 1111, 1113, 1115, 1117, 1119, 1121, 1123, 1125,1127, 1129, 1131, 1133, 1135, 1137, 1139, 1141, 1143, 1145, 1147, 1149,1151, 1153, 1155, 1157, 1159, 1161, 1163, 1165, 1167, 1169, 1171, 1173,1175, 1177, 1179, 1181, 1183, 1185, 1187, 1189, 1191, 1193, 1195, 1197,1199, 1201, 1203, 1205, 1207, 1209, 1211, 1213, 1215, 1217, 1219, 1221,1223, 1225, 1227, 1229, 1231, 1233, 1235, 1237, 1239, 1241, 1243, 1245,1247, 1249, 1251, 1253, 1255, 1257, 1259, 1261, 1263, 1265, 1267, 1269,1271, 1273, 1275, 1277, 1279, 1281, 1283, 1285, 1287, 1289, 1291, 1293,1295, 1297, 1299, 1301, 1303, 1305, 1307, 1309, 1310, 1311, 1313, 1315,1317, 1319, 1321, 1323, 1325, 1327, 1329, 1331, 1333, 1335, 1337, 1339,1341, 1343, 1345, 1347, 1349, 1351, 1353, 1355, 1357, 1359, 1361, 1363,1365, 1367, 1369, 1371, 1373, 1375, 1377, 1379, 1381, 1383, 1385, 1387,1389, 1391, 1393, 1395, 1397, 1399, 1401, 1403, 1405, 1407, 1409, 1411,1413, 1415, 1417, 1419, 1421, 1423, 1425, 1427, 1429, 1431, 1433, 1435,1437, 1439, 1441, 1443, 1445, 1447, 1449, 1451, 1453, 1455, 1457, 1459,1461, 1463, 1465, 1467, 1469, 1471, 1473, 1475, 1477, 1479, 1481, 1483,1485, 1487, 1489, 1491, 1493, 1495, 1497, 1499, 1501, 1503, 1505, 1507,1509, 1511, 1513, 1515, 1517, 1519, 1521, 1523, 1525, 1527, 1529, 1531,1533, 1535, 1537, 1539, 1541, 1543, 1545, 1547, 1549, 1551, 1553, 1555,1557, 1559, 1561, 1563, 1565, 1567, 1569, 1571, 1573, 1575, 1577, 1579,1581, 1583, 1585, 1587, 1589, 1591, 1593, 1595, 1597, 1599, 1601, 1603,1605, 1607, 1609, 1611, 1613, 1615, 1617, 1619, 1621, 1623, 1625, 1627,1629, 1631, 1633, 1635, 1637, 1639, 1641, 1643, 1645, 1647, 1649, 1651,1653, 1655, 1657, 1659, 1661, 1663, 1665, 1667, 1669, 1671, 1673, 1675,1677, 1679, 1681, 1683, 1685, 1687, 1689, 1691, 1693, 1695, 1697, 1699,1701, 1703, 1705, 1707, 1709, 1711, 1713, 1715, 1717, 1719, 1721, 1723,1725, 1727, 1729, 1731, 1733, 1735, 1737, 1739, 1741, 1743, 1745, 1747,1749, 1751, 1753, 1755, 1757, 1759, 1761, 1763, 1765, 1767, 1769, 1771,1773, 1775, 1777, 1779, 1781, 1783, 1785, 1787, 1789, 1791, 1793, 1795,1797, 1799, 1801, 1803, 1805, 1807, 1809, 1811, 1813, 1815, 1817, 1819,1821, 1823, 1825, 1827, 1829, 1831, 1833, 1835, 1837, 1839, 1841, 1843,1845, 1847, 1849, 1851, 1853, 1855, 1857, 1859, 1861, 1863, 1865, 1867,1869, 1871, 1873, 1875, 1877, 1879, 1881, 1883, 1885, 1887, 1889, 1891,1893, 1895, 1897, 1899, 1901, 1903, 1905, 1907, 1909, 1911, 1913, 1915,1917, 1919, 1921, 1923, 1925, 1927, 1929, 1931, 1933, 1935, 1937, 1939,1941, 1943, 1945, 1947, 1949, 1951, 1953, 1955, 1957, 1959, 1961, 1963,1965, 1967, 1969, 1971, 1973, 1975, 1977, 1979, 1981, 1983, 1985, 1987,1989, 1991, 1993, 1995, 1997, 1999, 2001, 2003, 2005, 2007, 2009, 2011,2013, 2015, 2017, 2019, 2021, 2023, 2025, 2027, 2029, 2031, 2033, 2035,2037, 2039, 2041, 2043, 2045, 2047, 2049, 2051, 2053, 2055, 2057, 2059,2061, 2063, 2065, 2067, 2069, 2071, 2073, 2075, 2077, 2079, 2081, 2083,2085, 2087, 2089, 2091, 2093, 2095, 2097, 2099, 2101, 2103, 2105, 2107,2109, 2111, 2113, 2115, 2117, 2119, 2121, 2123, 2125, 2127, 2129, 2131,2133, 2135, 2137, 2139, 2141, 2143, 2145, 2147, 2149, 2151, 2153, 2155,2157, 2159, 2161, 2163, 2165, 2167, 2169, 2171, 2173, 2175, 2177, 2179,2181, 2183, 2185, 2187, 2189, 2191, 2193, 2195, 2197, 2199, 2201, 2203,2205, 2207, 2209, 2211, 2213, 2215, 2217, 2219, 2221, 2223, 2225, 2227,2229, 2231, 2233, 2235, 2237, 2239, 2241, 2243, 2245, 2247, 2249, 2251,2253, 2255, 2257, 2259, 2261, 2263, 2265, 2267, 2269, 2271, 2273, 2275,2277, 2279, 2281, 2283, 2285, 2287, 2289, 2291, 2293, 2295, 2297, 2299,2301, 2303, 2305, 2307, 2309, 2311, 2313, 2315, 2317, 2319, 2321, 2323,2325, 2327, 2329, 2331, 2333, 2335, 2337, 2339, 2341, 2343, 2345, 2347,2349, 2351, 2353, 2355, 2357, 2359, 2361, 2363, 2365, 2367, 2369, 2371,2373, 2375, 2377, 2379, 2381, 2383, 2385, 2387, 2389, 2391, 2393, 2395,2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2413, 2415, 2417, 2419,2421, 2423, 2425, 2427, 2429, 2431, 2433, 2435, 2437, 2439, 2441, 2443,2445, 2447, 2449, 2451, 2453, 2455, 2457, 2459, 2461, 2463, 2465, 2467,2469, 2471, 2473, 2475, 2477, 2479, 2481, 2483, 2485, 2487, 2489, 2491,2493, 2495, 2497, 2499, 2501, 2503, 2505, 2507, 2509, 2511, 2513, 2515,2517, 2519, 2521, 2523, 2525, 2527, 2529, 2531, 2533, 2635, 2537, 2539,2541, 2543, 2545, 2547, 2549, 2551, 2553, 2555, 2557, 2559, 2561, 2563,2565, 2567, 2569, 2571, 2573, 2575, 2577, 2579, 2581, 2583, 2585, 2587,2589, 2591, 2593, 2595, 2597, 2599, 2601, 2603, 2605, 2607, 2609, 2611,2613, 2615, 2617, 2619, 2621, 2623, 2625, 2627, 2629, 2631, 2633, 2635,2637, 2639, 2641, 2643, 2645, 2647, 2649, 2651, 2653, 2655, 2657, 2659,2661, 2663, 2665, 2667, 2669, 2671, 2673, 2675, 2677, 2679, 2681, 2683,2685, 2687, 2689, 2691, 2693, 2695, 2697, 2699, 2701, 2703, 2705, 2707,2709, 2711, 2713, 2715, 2717, 2719, 2721, 2723, 2725, 2727, 2729, 2731,2733, 2735, 2737, 2739, 2741, 2743, 2745, 2747, 2749, 2751, 2753, 2755,2757, 2759, 2761, 2763, 2765, 2767, 2769, 2771, 2773, 2775, 2777, 2779,2781, 2783, 2785, 2787, 2789, 2791, 2793, 2795, 2797, 2799, 2801, 2803,2805, 2807, 2809, 2811, 2813, 2815, 2817, 2819, 2821, 2823, 2825, 2827,2829, 2831, 2833, 2835, 2837, 2839, 2841, 2843, 2845, 2847, andcomplementary sequences thereof, wherein the encoded HHDH polypeptidehas greater HHDH activity than the wildtype HHDH of SEQ ID NO: 2.Typically, these HHDH polypeptides have HHDH activity that is either:(a) at least 1.4-fold greater than the HHDH polypeptide of SEQ ID NO: 2,as measured in the assay of Example 5A; or (b) at least 1.5-fold greaterthan the HHDH polypeptide of SEQ ID NO: 730, as measured in the assay ofExample 5D.

Some of the HHDH polypeptides described in part (b) of the previousparagraph have at least about 2 fold greater HHDH activity than the HHDHpolypeptide corresponding to SEQ ID NO: 730, some at least 2.5-fold,3-fold, 4-fold, up to about 10-fold or 100-fold greater HHDH activitythan the HHDH polypeptide corresponding to SEQ ID NO: 730.

HHDH polypeptides described in part (b) in paragraph 0125 include thosehaving amino acid sequences that comprise one or more amino acidresidues selected from the group consisting of Q at position 38, I atposition 52, L at position 70, A at position 82, P at position 83, V atposition 84, W at position 86, R at position 87, D at position 99, T atposition 100, A at position 174, S at position 189, S at position 195, Rat position 203, and T at position 222, wherein amino acid position isdetermined by optimal alignment of the amino acid sequence of the HHDHpolypeptide with SEQ ID NO: 2, These HHDH polypeptides may have anycombination of two or more, three or more, four or more, five or more,six or more, seven or more, eight or more, nine or more, ten or more,eleven or more, twelve or more, thirteen or more, fourteen or more, orall fifteen of these amino acid residues (at the specified position) intheir sequences. In certain embodiments, these HHDH polypeptidescomprise one or more amino acid residues selected from the groupconsisting of Q at position 38, I at position 52, L at position 70, A atposition 82, P at position 83, V at position 84, W at position 86, D atposition 99, T at position 100, A at position 174, S at position 189, Sat position 195, and R at position 203. These HHDH polypeptides may haveany combination of two or more, three or more, four or more, five ormore, six or more, seven or more, eight or more, nine or more, ten ormore, eleven or more, twelve or more, or all thirteen of these aminoacid residues (at the specified position) in their sequences.

Amino acid residue position of the encoded polypeptides described inparagraph 0125 is determined by optimal alignment with a reference aminoacid sequence. The reference amino acid sequence is encoded by thereference nucleic acid sequence and thus selected from the groupconsisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98,100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126,128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154,156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182,184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210,212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238,240, 242, 244, 246, 248, 250, 252, 254, 256, 258. 260, 262, 264, 266,268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294,296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322,324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350,352, 354, 356, 358, 360, 362, 364, 368, 370, 372, 374, 376, 378, 380,382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408,410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436,438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464,466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492,494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520,522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548,550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576,578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604,606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632,634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660,662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688,690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716,718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744,746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772,774, 776, 778, 780, 782, 784, 786, 788, 790, 792, 794, 796, 798, 800,802, 804, 806, 808, 810, 812, 814, 816, 818, 820, 822, 824, 826, 828,830, 832, 834, 836, 838, 840, 842, 844, 846, 848, 850, 852, 854, 856,858, 860, 862, 864, 866, 868, 870, 872, 874, 876, 878, 880, 882, 884,886, 888, 890, 892, 894, 896, 898, 900, 902, 904, 906, 908, 910, 912,914, 916, 918, 920, 922, 924, 926, 928, 930, 932, 934, 936, 938, 940,942, 944, 946, 948, 950, 952, 954, 956, 958, 960, 962, 964, 966, 968,970, 972, 974, 976, 978, 980, 982, 984, 986, 988, 990, 992, 994, 996,998, 1000, 1002, 1004, 1006, 1008, 1010, 1012, 1014, 1016, 1018, 1020,1022, 1024, 1026, 1028, 1030, 1032, 1034, 1036, 1038, 1040, 1042, 1044,1046, 1048, 1050, 1052, 1054, 1056, 1058, 1060, 1062, 1064, 1066, 1068,1070, 1072, 1074, 1076, 1078, 1080, 1082, 1084, 1086, 1088, 1090, 1092,1094, 1096, 1098, 1100, 1102, 1104, 1106, 1108, 1110, 1112, 1114, 1116,1118, 1120, 1122, 1124, 1126, 1128, 1130, 1132, 1134, 1136, 1138, 1140,1142, 1144, 1146, 1148, 1150, 1152, 1154, 1156, 1158, 1160, 1162, 1164,1166, 1168, 1170, 1172, 1174, 1176, 1178, 1180, 1182, 1184, 1186, 1188,1190, 1192, 1194, 1196, 1198, 1200, 1202, 1204, 1206, 1208, 1210, 1212,1214, 1216, 1218, 1220, 1222, 1224, 1226, 1228, 1230, 1232, 1234, 1236,1238, 1240, 1242, 1244, 1246, 1248, 1250, 1252, 1254, 1256, 1258, 1260,1262, 1264, 1266, 1268, 1270, 1272, 1274, 1276, 1278, 1280, 1282, 1284,1286, 1288, 1290, 1292, 1294, 1296, 1298, 1300, 1302, 1304, 1306, 1308,1310, 1312, 1314, 1316, 1318, 1320, 1322, 1324, 1326, 1328, 1330, 1332,1334, 1336, 1338, 1340, 1342, 1344, 1346, 1348, 1350, 1352, 1354, 1356,1358, 1360, 1362, 1364, 1366, 1368, 1370, 1372, 1374, 1376, 1378, 1380,1382, 1384, 1386, 1388, 1390, 1392, 1394, 1396, 1398, 1400, 1402, 1404,1406, 1408, 1410, 1412, 1414, 1416, 1418, 1420, 1422, 1424, 1426, 1428,1430, 1432, 1434, 1436, 1438, 1440, 1442, 1444, 1446, 1448, 1450, 1452,1454, 1456, 1458, 1460, 1462, 1464, 1466, 1468, 1470, 1472, 1474, 1476,1478, 1480, 1482, 1484, 1486, 1488, 1490, 1492, 1494, 1496, 1498, 1500,1502, 1504, 1506, 1508, 1510, 1512, 1514, 1516, 1518, 1520, 1522, 1524,1526, 1528, 1530, 1532, 1534, 1536, 1538, 1540, 1542, 1544, 1546, 1548,1550, 1552, 1554, 1556, 1558, 1560, 1562, 1564, 1566, 1568, 1570, 1572,1574, 1576, 1578, 1580, 1582, 1584, 1586, 1588, 1590, 1592, 1594, 1596,1598, 1600, 1602, 1604, 1606, 1608, 1610, 1612, 1612, 1614, 1616, 1618,1620, 1622, 1624, 1626, 1628, 1630, 1632, 1634, 1636, 1638, 1640, 1642,1644, 1646, 1648, 1650, 1652, 1654, 1656, 1658, 1660, 1662, 1664, 1666,1668, 1670, 1672, 1674, 1676, 1678, 1680, 1682, 1684, 1686, 1688, 1690,1692, 1694, 1696, 1698, 1700, 1702, 1704, 1706, 1708, 1710, 1712, 1714,1716, 1718, 1720, 1722, 1724, 1726, 1728, 1730, 1732, 1734, 1736, 1738,1740, 1742, 1744, 1746, 1748, 1750, 1752, 1754, 1756, 1758, 1760, 1762,1764, 1766, 1768, 1770, 1772, 1774, 1776, 1778, 1780, 1782, 1784, 1786,1788, 1790, 1792, 1794, 1796, 1798, 1800, 1802, 1804, 1806, 1808, 1810,1812, 1814, 1816, 1818, 1820, 1822, 1824, 1826, 1828, 1830, 1832, 1834,1836, 1838, 1840, 1842, 1844, 1846, 1848, 1850, 1852, 1854, 1856, 1858,1860, 1862, 1864, 1866, 1868, 1870, 1872, 1874, 1876, 1878, 1880, 1882,1884, 1886, 1888, 1890, 1892, 1894, 1896, 1898, 1900, 1902, 1904, 1906,1908, 1910, 1912, 1914, 1916, 1918, 1920, 1922, 1924, 1926, 1928, 1930,1932, 1934, 1936, 1938, 1940, 1942, 1944, 1946, 1948, 1950, 1952, 1954,1956, 1958, 1960, 1962, 1964, 1966, 1968, 1970, 1972, 1974, 1976, 1978,1980, 1982, 1984, 1986, 1988, 1990, 1992, 1994, 1996, 1998, 2000, 2002,2004, 2006, 2008, 2010, 2012, 2014, 2016, 2018, 2020, 2022, 2024, 2026,2028, 2030, 2032, 2034, 2036, 2038, 2040, 2042, 2044, 2046, 2048, 2050,2052, 2054, 2056, 2058, 2060, 2062, 2064, 2066, 2068, 2070, 2072, 2074,2076, 2078, 2080, 2082, 2084, 2086, 2088, 2090, 2092, 2094, 2096, 2098,2100, 2102, 2104, 2106, 2108, 2110, 2112, 2114, 2116, 2118, 2120, 2122,2124, 2126, 2128, 2130, 2132, 2134, 2136, 2138, 2140, 2142, 2144, 2146,2148, 2150, 2152, 2154, 2156, 2158, 2160, 2162, 2164, 2166, 2168, 2170,2172, 2174, 2176, 2178, 2180, 2182, 2184, 2186, 2188, 2190, 2192, 2194,2196, 2198, 2200, 2202, 2204, 2206, 2208, 2210, 2212, 2214, 2216, 2218,2220, 2222, 2224, 2226, 2228, 2230, 2232, 2234, 2236, 2238, 2240, 2242,2244, 2246, 2248, 2250, 2252, 2254, 2256, 2258, 2260, 2262, 2264, 2266,2268, 2270, 2272, 2274, 2276, 2278, 2280, 2282, 2284, 2286, 2288, 2290,2292, 2294, 2296, 2298, 2300, 2302, 2304, 2306, 2308, 2310, 2312, 2316,2318, 2320, 2322, 2324, 2326, 2328, 2330, 2332, 2334, 2336, 2338, 2340,2342, 2344, 2346, 2348, 2350, 2352, 2354, 2356, 2358, 2360, 2362, 2364,2366, 2368, 2370, 2372, 2374, 2376, 2378, 2380, 2382, 2384, 2386, 2388,2390, 2392, 2394, 2396, 2398, 2400, 2402, 2404, 2406, 2408, 2410, 2412,2414, 2416, 2418, 2420, 2422, 2424, 2426, 2428, 2430, 2432, 2434, 2436,2438, 2440, 2442, 2444, 2446, 2448, 2450, 2452, 2454, 2456, 2458, 2460,2462, 2464, 2466, 2468, 2470, 2472, 2474, 2476, 2478, 2480, 2482, 2484,2486, 2488, 2490, 2492, 2494, 2496, 2498, 2500, 2502, 2504, 2506, 2508,2510, 2512, 2514, 2516, 2518, 2520, 2522, 2524, 2526, 2528, 2530, 2532,2534, 2536, 2538, 2540, 2542, 2544, 2546, 2548, 2550, 2552, 2554, 2556,2558, 2560, 2562, 2564, 2566, 2568, 2570, 2572, 2574, 2576, 2578, 2580,2582, 2584, 2586, 2588, 2590, 2592, 2594, 2596, 2598, 2600, 2602, 2604,2606, 2608, 2610, 2612, 2614, 2616, 2618, 2620, 2622, 2624, 2626, 2628,2630, 2632, 2634, 2638, 2640, 2642, 2644, 2646, 2648, 2650, 2652, 2654,2656, 2658, 2660, 2662, 2664, 2666, 2668, 2670, 2672, 2674, 2676, 2678,2680, 2682, 2684, 2686, 2688, 2690, 2692, 2694, 2696, 2698, 2700, 2702,2704, 2706, 2708, 2710, 2712, 2714, 2716, 2718, 2720, 2722, 2724, 2726,2728, 2730, 2732, 2734, 2736, 2738, 2740, 2742, 2744, 2746, 2748, 2750,2752, 2754, 2756, 2758, 2760, 2762, 2764, 2766, 2768, 2770, 2772, 2774,2776, 2778, 2780, 2782, 2784, 2786, 2788, 2790, 2792, 2794, 2796, 2798,2800, 2802, 2804, 2806, 2808, 2810, 2812, 2814, 2816, 2818, 2820, 2822,2824, 2826, 2828, 2830, 2832, 2834, 2836, 2838, 2840, 2842, 2844, 2846,and 2848, respectively.

In a further embodiment, the present invention provides HHDHpolypeptides that exhibit HHDH activity in the presence of productcyanohydrin, e.g., ethyl (R) 4-cyano-3-hydroxybutyrate. HHDHpolypeptides that are resistant to inhibition by cyanohydrin may bereadily identified using the assay described in Example 5B. Example 5Bdescribes a protocol for assaying for enzymes that are robust withrespect to product inhibition. Exemplary HHDH polypeptides that exhibitsignificant HHDH activity even in the presence of product, ethyl(R)-4-cyano-3-hydroxybutyrate in the assay described in Example 5Binclude the HHDH polypeptides corresponding to SEQ ID NOS: 98, 100, 102,104, 106, 108, 120, 122, 124, 126, 128, 130, 132, 136, 138, 140, 142,144, 146, 148, 150, 152, 160, 174, 176, 178, 188, 190, 192, 194, 196,198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224,226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252,254, 256, 258, 260, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294,296, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324,326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352,354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380,382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408,410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436,438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464,466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492,494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520,522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548,550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576,578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604,606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632,634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660,662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688,690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716,718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744,746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772,774, 776, 778, 780, 782, 784, 786, 788, 790, 792, 794, 796, 798, 800,802, 804, 806, 808, 810, 812, 814, 816, 818, 820, 822, 824, 826, 828,830, 832, 834, 836, 838, 840, 842, 844, 846, 848, 850, 852, 854, 856,858, 860, 862, 864, 866, 868, 870, 872, 874, 876, 878, 880, 882, 884,886, 888, 890, 892, 894, 896, 898, 900, 902, 904, 906, 908, 910, 912,914, 916, 918, 920, 922, 924, 926, 928, 930, 932, 934, 936, 938, 940,942, 944, 946, 948, 950, 952, 954, 956, 958, 960, 962, 964, 966, 968,970, 972, 974, 976, 978, 980, 982, 984, 986, 988, 990, 992, 994, 996,998, 1000, 1002, 1004, 1006, 1008, 1010, 1012, 1014, 1016, 1018, 1020,1022, 1024, 1026, 1028, 1030, 1032, 1034, 1036, 1038, 1040, 1042, 1044,1046, 1048, 1050, 1052, 1054, 1056, 1058, 1060, 1062, 1064, 1066, 1068,1070, 1072, 1074, 1076, 1078, 1080, 1082, 1084, 1086, 1088, 1090, 1092,1094, 1096, 1098, 1100, 1102, 1104, 1106, 1108, 1110, 1112, 1114, 1116,1118, 1120, 1122, 1124, 1126, 1128, 1130, 1132, 1134, 1136, 1138, 1140,1142, 1144, 1144, 1146, 1148, 1150, 1152, 1154, 1156, 1158, 1160, 1162,1164, 1166, 1168, 1170, 1172, 1174, 1176, 1178, 1180, 1182, 1184, 1186,1188, 1190, 1192, 1194, 1196, 1198, 1200, 1202, 1204, 1206, 1208, 1210,1212, 1214, 1216, 1218, 1220, 1222, 1224, 1226, 1228, 1230, 1232, 1234,1236, 1238, 1240, 1242, 1244, 1246, 1248, 1250, 1252, 1254, 1256, 1258,1260, 1262, 1264, 1266, 1268, 1270, 1272, 1274, 1276, 1278, 1280, 1282,1284, 1286, 1288, 1290, 1292, 1294, 1296, 1298, 1300, 1302, 1304, 1306,1308, 1310, 1312, 1314, 1316, 1318, 1320, 1322, 1324, 1326, 1328, 1330,1332, 1334, 1336, 1338, 1340, 1342, 1344, 1346, 1348, 1350, 1352, 1354,1356, 1358, 1360, 1362, 1364, 1366, 1368, 1370, 1372, 1374, 1376, 1378,1380, 1382, 1384, 1386, 1388, 1390, 1392, 1394, 1396, 1398, 1400, 1402,1404, 1406, 1408, 1410, 1412, 1414, 1416, 1418, 1420, 1422, 1424, 1426,1428, 1430, 1432, 1434, 1436, 1438, 1440, 1442, 1444, 1446, 1448, 1450,1452, 1454, 1456, 1458, 1460, 1462, 1464, 1466, 1468, 1470, 1472, 1474,1476, 1478, 1480, 1482, 1484, 1486, 1488, 1490, 1492, 1494, 1496, 1498,1500, 1502, 1504, 1506, 1508, 1510, 1512, 1514, 1516, 1518, 1520, 1522,1524, 1526, 1528, 1530, 1532, 1534, 1536, 1538, 1540, 1542, 1544, 1546,1548, 1550, 1552, 1554, 1556, 1558, 1560, 1562, 1564, 1566, 1568, 1570,1572, 1574, 1576, 1578, 1580, 1582, 1584, 1586, 1588, 1590, 1592, 1594,1596, 1598, 1600, 1602, 1604, 1606, 1608, 1610, 1612, 1612, 1614, 1616,1618, 1620, 1622, 1624, 1626, 1628, 1630, 1632, 1634, 1636, 1638, 1640,1642, 1644, 1646, 1648, 1650, 1652, 1654, 1656, 1658, 1660, 1662, 1664,1666, 1668, 1670, 1672, 1674, 1676, 1678, 1680, 1682, 1684, 1686, 1688,1690, 1692, 1694, 1696, 1698, 1700, 1702, 1704, 1706, 1708, 1710, 1712,1714, 1716, 1718, 1720, 1722, 1724, 1726, 1728, 1730, 1732, 1734, 1736,1738, 1740, 1742, 1744, 1746, 1748, 1750, 1752, 1754, 1756, 1758, 1760,1762, 1764, 1766, 1768, 1770, 1772, 1774, 1776, 1778, 1780, 1782, 1784,1786, 1788, 1790, 1792, 1794, 1796, 1798, 1800, 1802, 1804, 1806, 1808,1810, 1812, 1814, 1816, 1818, 1820, 1822, 1824, 1826, 1828, 1830, 1832,1834, 1836, 1838, 1840, 1842, 1844, 1846, 1848, 1850, 1852, 1854, 1856,1858, 1860, 1862, 1864, 1866, 1868, 1870, 1872, 1874, 1876, 1878, 1880,1882, 1884, 1886, 1888, 1890, 1892, 1894, 1896, 1898, 1900, 1902, 1904,1906, 1908, 1910, 1912, 1914, 1916, 1918, 1920, 1922, 1924, 1926, 1928,1930, 1932, 1934, 1936, 1938, 1940, 1942, 1944, 1946, 1948, 1950, 1952,1954, 1956, 1958, 1960, 1962, 1964, 1966, 1968, 1970, 1972, 1974, 1976,1978, 1980, 1982, 1984, 1986, 1988, 1990, 1992, 1994, 1996, 1998, 2000,2002, 2004, 2006, 2008, 2010, 2012, 2014, 2016, 2018, 2020, 2022, 2024,2026, 2028, 2030, 2032, 2034, 2036, 2038, 2040, 2042, 2044, 2046, 2048,2050, 2052, 2054, 2056, 2058, 2060, 2062, 2064, 2066, 2068, 2070, 2072,2074, 2076, 2078, 2080, 2082, 2084, 2086, 2088, 2090, 2092, 2094, 2096,2098, 2100, 2102, 2104, 2106, 2108, 2110, 2112, 2114, 2116, 2118, 2120,2122, 2124, 2126, 2128, 2130, 2132, 2134, 2136, 2138, 2140, 2142, 2144,2146, 2148, 2150, 2152, 2154, 2156, 2158, 2160, 2162, 2164, 2166, 2168,2170, 2172, 2174, 2176, 2178, 2180, 2182, 2184, 2186, 2188, 2190, 2192,2194, 2196, 2198, 2200, 2202, 2204, 2206, 2208, 2210, 2212, 2214, 2216,2218, 2220, 2222, 2224, 2226, 2228, 2230, 2232, 2234, 2236, 2238, 2240,2242, 2244, 2246, 2248, 2250, 2252, 2254, 2256, 2258, 2260, 2262, 2264,2266, 2268, 2270, 2272, 2274, 2276, 2278, 2280, 2282, 2284, 2286, 2288,2290, 2292, 2294, 2296, 2298, 2300, 2302, 2304, 2306, 2308, 2310, 2312,2316, 2318, 2320, 2322, 2324, 2326, 2328, 2330, 2332, 2334, 2336, 2338,2340, 2342, 2344, 2346, 2348, 2350, 2352, 2354, 2356, 2358, 2360, 2362,2364, 2366, 2368, 2370, 2372, 2374, 2376, 2378, 2380, 2382, 2384, 2386,2388, 2390, 2392, 2394, 2396, 2398, 2400, 2402, 2404, 2406, 2408, 2410,2412, 2414, 2416, 2418, 2420, 2422, 2424, 2426, 2428, 2430, 2432, 2434,2436, 2438, 2440, 2442, 2444, 2446, 2448, 2450, 2452, 2454, 2456, 2458,2460, 2462, 2464, 2466, 2468, 2470, 2472, 2474, 2476, 2478, 2480, 2482,2484, 2486, 2488, 2490, 2492, 2494, 2496, 2498, 2500, 2502, 2504, 2506,2508, 2510, 2512, 2514, 2516, 2518, 2520, 2522, 2524, 2526, 2528, 2530,2532, 2534, 2536, 2538, 2540, 2542, 2544, 2546, 2548, 2550, 2552, 2554,2556, 2558, 2560, 2562, 2564, 2566, 2568, 2570, 2572, 2574, 2576, 2578,2580, 2582, 2584, 2586, 2588, 2590, 2592, 2594, 2596, 2598, 2600, 2602,2604, 2606, 2608, 2610, 2612, 2614, 2616, 2618, 2620, 2622, 2624, 2626,2628, 2630, 2632, 2634, 2638, 2640, 2642, 2644, 2646, 2648, 2650, 2652,2654, 2656, 2658, 2660, 2662, 2664, 2666, 2668, 2670, 2672, 2674, 2676,2678, 2680, 2682, 2684, 2686, 2688, 2690, 2692, 2694, 2696, 2698, 2700,2702, 2704, 2706, 2708, 2710, 2712, 2714, 2716, 2718, 2720, 2722, 2724,2726, 2728, 2730, 2732, 2734, 2736, 2738, 2740, 2742, 2744, 2746, 2748,2750, 2752, 2754, 2756, 2758, 2760, 2762, 2764, 2766, 2768, 2770, 2772,2774, 2776, 2778, 2780, 2782, 2784, 2786, 2788, 2790, 2792, 2794, 2796,2798, 2800, 2802, 2804, 2806, 2808, 2810, 2812, 2814, 2816, 2818, 2820,2822, 2824, 2826, 2828, 2830, 2832, 2834, 2836, 2838, 2840, 2842, 2844,2846, and 2848.

Polypeptides that exhibit the ability to convert ethyl(S)-4-chloro-3-hydroxybutyrate to ethyl (R)-4-cyano-3-hydroxybutyrate inthe assay of Example 5B also demonstrate HHDH activity in the assay ofExample 5A. All of the polypeptide sequences set forth in the SequenceListing exhibit activity in the assay of Example 5A.

Ethyl-4-chloroacetoacetate (ECAA) is the substrate for the coupledreduction reaction using ketoreductase (KRED)/glucose dehydrogenase(GDH) to produce ethyl (S)-4-chloro-3-hydroxybutyrate (ECHB). See WO2004/15132 and WO 2005/018579, both incorporated herein by reference.The ECHB is then used as substrate for the HHDH reaction. However, theECAA starting material is a potent inhibitor (K_(i) approximately=70 μM)of HHDH. Because the KRED/GDH catalyzed reaction may go to 99.9%completion, instead of the desired 99.97%, 0.1% ECAA may remain in theECHB material. This 0.1% ECAA can inhibit the HHDH reaction. In otherwords, the remaining substrate from the first reaction is an inhibitorin the second reaction. Hence, it is desirable that the HHDHpolypeptides of the present invention have resistance to inhibition byECAA.

Applicants have discovered that they could make the following residuechanges relative to the alignment in SEQ ID NO: 2 to produce HHDHpolypeptides of the present invention that demonstrate increasedresistance against inhibition by ECAA: A4V, A82Y, A134V, G136W, G136V,L142R, L178V, W238L, A240T, W249Y, M252I, K10L, E181I/K, H198N, K215E,S180I/D/T, W139H, and M54V. Thus, in another embodiment, the presentinvention is directed to an HHDH polypeptide of the present inventionthat is resistant to inhibition by ECAA, and having an amino acidsequence with one or more amino acid residues selected from the groupconsisting of V at position 4, Y at position 82, V at position 134, W atposition 136, V at position 136, R at position 142, V at position 178, Lat position 238, T at position 240, Y at position 249, I at position252, L at position 10, I or K at position 181, N at position 198, E atposition 215, I or D or T at position 280, H at position 139, and V atposition 54, wherein amino acid position is determined by alignment ofthe amino acid sequence of the HHDH polypeptide with SEQ ID NO:2. Insome embodiments, HHDH polypeptides that are resistant to inhibition byECAA have an amino acid sequence with one or more amino acid residuesare selected from the group consisting of L at position 10, I or K atposition 181, N at position 198, E at position 215, D or T at position180, H at position 139, and V at position 54. Specific HHDH polypeptidesthat are resistant to inhibition by ECAA have an amino acid sequencewith one or more amino acid residues selected from the group consistingof D or T at position 180 and K at position 181. A method for testingthe HHDH polypeptides of the present invention for their activity in thepresence of ECAA is disclosed in Example 5C herein. A gaschromatographic method for screening the reaction products from Example5C, and determining the amount of product produced, is disclosed inExample 6B herein.

Specific HHDH polypeptides of the present invention include those havingan amino acid sequence corresponding to SEQ ID NOS: 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88,90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118,120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146,148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174,176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202,204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230,232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258.260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286,288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314,316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342,344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 368, 370, 372,374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400,402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428,430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456,458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484,486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512,514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540,542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568,570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596,598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624,626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652,654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680,682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708,710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736,738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764,766, 768, 770, 772, 774, 776, 778, 780, 782, 784, 786, 788, 790, 792,794, 796, 798, 800, 802, 804, 806, 808, 810, 812, 814, 816, 818, 820,822, 824, 826, 828, 830, 832, 834, 836, 838, 840, 842, 844, 846, 848,850, 852, 854, 856, 858, 860, 862, 864, 866, 868, 870, 872, 874, 876,878, 880, 882, 884, 886, 888, 890, 892, 894, 896, 898, 900, 902, 904,906, 908, 910, 912, 914, 916, 918, 920, 922, 924, 926, 928, 930, 932,934, 936, 938, 940, 942, 944, 946, 948, 950, 952, 954, 956, 958, 960,962, 964, 966, 968, 970, 972, 974, 976, 978, 980, 982, 984, 986, 988,990, 992, 994, 996, 998, 1000, 1002, 1004, 1006, 1008, 1010, 1012, 1014,1016, 1018, 1020, 1022, 1024, 1026, 1028, 1030, 1032, 1034, 1036, 1038,1040, 1042, 1044, 1046, 1048, 1050, 1052, 1054, 1056, 1058, 1060, 1062,1064, 1066, 1068, 1070, 1072, 1074, 1076, 1078, 1080, 1082, 1084, 1086,1088, 1090, 1092, 1094, 1096, 1098, 1100, 1102, 1104, 1106, 1108, 1110,1112, 1114, 1116, 1118, 1120, 1122, 1124, 1126, 1128, 1130, 1132, 1134,1136, 1138, 1140, 1142, 1144, 1146, 1148, 1150, 1152, 1154, 1156, 1158,1160, 1162, 1164, 1166, 1168, 1170, 1172, 1174, 1176, 1178, 1180, 1182,1184, 1186, 1188, 1190, 1192, 1194, 1196, 1198, 1200, 1202, 1204, 1206,1208, 1210, 1212, 1214, 1216, 1218, 1220, 1222, 1224, 1226, 1228, 1230,1232, 1234, 1236, 1238, 1240, 1242, 1244, 1246, 1248, 1250, 1252, 1254,1256, 1258, 1260, 1262, 1264, 1266, 1268, 1270, 1272, 1274, 1276, 1278,1280, 1282, 1284, 1286, 1288, 1290, 1292, 1294, 1296, 1298, 1300, 1302,1304, 1306, 1308, 1310, 1312, 1314, 1316, 1318, 1320, 1322, 1324, 1326,1328, 1330, 1332, 1334, 1336, 1338, 1340, 1342, 1344, 1346, 1348, 1350,1352, 1354, 1356, 1358, 1360, 1362, 1364, 1366, 1368, 1370, 1372, 1374,1376, 1378, 1380, 1382, 1384, 1386, 1388, 1390, 1392, 1394, 1396, 1398,1400, 1402, 1404, 1406, 1408, 1410, 1412, 1414, 1416, 1418, 1420, 1422,1424, 1426, 1428, 1430, 1432, 1434, 1436, 1438, 1440, 1442, 1444, 1446,1448, 1450, 1452, 1454, 1456, 1458, 1460, 1462, 1464, 1466, 1468, 1470,1472, 1474, 1476, 1478, 1480, 1482, 1484, 1486, 1488, 1490, 1492, 1494,1496, 1498, 1500, 1502, 1504, 1506, 1508, 1510, 1512, 1514, 1516, 1518,1520, 1522, 1524, 1526, 1528, 1530, 1532, 1534, 1536, 1538, 1540, 1542,1544, 1546, 1548, 1550, 1552, 1554, 1556, 1558, 1560, 1562, 1564, 1566,1568, 1570, 1572, 1574, 1576, 1578, 1580, 1582, 1584, 1586, 1588, 1590,1592, 1594, 1596, 1598, 1600, 1602, 1604, 1606, 1608, 1610, 1612, 1612,1614, 1616, 1618, 1620, 1622, 1624, 1626, 1628, 1630, 1632, 1634, 1636,1638, 1640, 1642, 1644, 1646, 1648, 1650, 1652, 1654, 1656, 1658, 1660,1662, 1664, 1666, 1668, 1670, 1672, 1674, 1676, 1678, 1680, 1682, 1684,1686, 1688, 1690, 1692, 1694, 1696, 1698, 1700, 1702, 1704, 1706, 1708,1710, 1712, 1714, 1716, 1718, 1720, 1722, 1724, 1726, 1728, 1730, 1732,1734, 1736, 1738, 1740, 1742, 1744, 1746, 1748, 1750, 1752, 1754, 1756,1758, 1760, 1762, 1764, 1766, 1768, 1770, 1772, 1774, 1776, 1778, 1780,1782, 1784, 1786, 1788, 1790, 1792, 1794, 1796, 1798, 1800, 1802, 1804,1806, 1808, 1810, 1812, 1814, 1816, 1818, 1820, 1822, 1824, 1826, 1828,1830, 1832, 1834, 1836, 1838, 1840, 1842, 1844, 1846, 1848, 1850, 1852,1854, 1856, 1858, 1860, 1862, 1864, 1866, 1868, 1870, 1872, 1874, 1876,1878, 1880, 1882, 1884, 1886, 1888, 1890, 1892, 1894, 1896, 1898, 1900,1902, 1904, 1906, 1908, 1910, 1912, 1914, 1916, 1918, 1920, 1922, 1924,1926, 1928, 1930, 1932, 1934, 1936, 1938, 1940, 1942, 1944, 1946, 1948,1950, 1952, 1954, 1956, 1958, 1960, 1962, 1964, 1966, 1968, 1970, 1972,1974, 1976, 1978, 1980, 1982, 1984, 1986, 1988, 1990, 1992, 1994, 1996,1998, 2000, 2002, 2004, 2006, 2008, 2010, 2012, 2014, 2016, 2018, 2020,2022, 2024, 2026, 2028, 2030, 2032, 2034, 2036, 2038, 2040, 2042, 2044,2046, 2048, 2050, 2052, 2054, 2056, 2058, 2060, 2062, 2064, 2066, 2068,2070, 2072, 2074, 2076, 2078, 2080, 2082, 2084, 2086, 2088, 2090, 2092,2094, 2096, 2098, 2100, 2102, 2104, 2106, 2108, 2110, 2112, 2114, 2116,2118, 2120, 2122, 2124, 2126, 2128, 2130, 2132, 2134, 2136, 2138, 2140,2142, 2144, 2146, 2148, 2150, 2152, 2154, 2156, 2158, 2160, 2162, 2164,2166, 2168, 2170, 2172, 2174, 2176, 2178, 2180, 2182, 2184, 2186, 2188,2190, 2192, 2194, 2196, 2198, 2200, 2202, 2204, 2206, 2208, 2210, 2212,2214, 2216, 2218, 2220, 2222, 2224, 2226, 2228, 2230, 2232, 2234, 2236,2238, 2240, 2242, 2244, 2246, 2248, 2250, 2252, 2254, 2256, 2258, 2260,2262, 2264, 2266, 2268, 2270, 2272, 2274, 2276, 2278, 2280, 2282, 2284,2286, 2288, 2290, 2292, 2294, 2296, 2298, 2300, 2302, 2304, 2306, 2308,2310, 2312, 2316, 2318, 2320, 2322, 2324, 2326, 2328, 2330, 2332, 2334,2336, 2338, 2340, 2342, 2344, 2346, 2348, 2350, 2352, 2354, 2356, 2358,2360, 2362, 2364, 2366, 2368, 2370, 2372, 2374, 2376, 2378, 2380, 2382,2384, 2386, 2388, 2390, 2392, 2394, 2396, 2398, 2400, 2402, 2404, 2406,2408, 2410, 2412, 2414, 2416, 2418, 2420, 2422, 2424, 2426, 2428, 2430,2432, 2434, 2436, 2438, 2440, 2442, 2444, 2446, 2448, 2450, 2452, 2454,2456, 2458, 2460, 2462, 2464, 2466, 2468, 2470, 2472, 2474, 2476, 2478,2480, 2482, 2484, 2486, 2488, 2490, 2492, 2494, 2496, 2498, 2500, 2502,2504, 2506, 2508, 2510, 2512, 2514, 2516, 2518, 2520, 2522, 2524, 2526,2528, 2530, 2532, 2534, 2536, 2538, 2540, 2542, 2544, 2546, 2548, 2550,2552, 2554, 2556, 2558, 2560, 2562, 2564, 2566, 2568, 2570, 2572, 2574,2576, 2578, 2580, 2582, 2584, 2586, 2588, 2590, 2592, 2594, 2596, 2598,2600, 2602, 2604, 2606, 2608, 2610, 2612, 2614, 2616, 2618, 2620, 2622,2624, 2626, 2628, 2630, 2632, 2634, 2638, 2640, 2642, 2644, 2646, 2648,2650, 2652, 2654, 2656, 2658, 2660, 2662, 2664, 2666, 2668, 2670, 2672,2674, 2676, 2678, 2680, 2682, 2684, 2686, 2688, 2690, 2692, 2694, 2696,2698, 2700, 2702, 2704, 2706, 2708, 2710, 2712, 2714, 2716, 2718, 2720,2722, 2724, 2726, 2728, 2730, 2732, 2734, 2736, 2738, 2740, 2742, 2744,2746, 2748, 2750, 2752, 2754, 2756, 2758, 2760, 2762, 2764, 2766, 2768,2770, 2772, 2774, 2776, 2778, 2780, 2782, 2784, 2786, 2788, 2790, 2792,2794, 2796, 2798, 2800, 2802, 2804, 2806, 2808, 2810, 2812, 2814, 2816,2818, 2820, 2822, 2824, 2826, 2828, 2830, 2832, 2834, 2836, 2838, 2840,2842, 2844, 2846, or 2848. All of these HHDH polypeptides havedemonstrated activity in at least one of the assays described in Example5A, 5B or 5D.

Exemplary HHDH polynucleotides that encode these HHDH polypeptides areprovided herein as SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,25, 27, 29, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61,63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97,99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125,127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153,155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181,183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209,211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237,239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265,267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293,295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321,323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349,351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377,379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405,407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433,435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461,463, 465, 467, 469, 4712, 473, 475, 477, 479, 481, 483, 485, 487, 489,491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517,519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545,547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573,575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601,603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629,631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657,659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685,687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713,715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 739, 741, 743,745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771,773, 775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799,801, 803, 805, 807, 809, 811, 813, 815, 817, 819, 821, 823, 825, 827,829, 831, 833, 835, 837, 839, 841, 843, 845, 847, 849, 851, 853, 855,857, 859, 861, 863, 865, 867, 869, 871, 873, 875, 877, 879, 881, 883,885, 887, 889, 891, 893, 895, 897, 899, 901, 903, 905, 907, 909, 911,913, 915, 917, 919, 921, 923, 925, 927, 929, 931, 933, 935, 937, 939,941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967,969, 971, 973, 975, 977, 979, 981, 983, 985, 987, 989, 991, 993, 995,997, 999, 1001, 1103, 1005, 1007, 1009, 1011, 1013, 1015, 1017, 1019,1021, 1023, 1025, 1027, 1029, 1031, 1033, 1035, 1037, 1039, 1041, 1043,1045, 1047, 1049, 1051, 1053, 1055, 1057, 1059, 1061, 1063, 1065, 1067,1069, 1071, 1073, 1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091,1093, 1095, 1097, 1099, 1101, 1103, 1105, 1107, 1109, 1111, 1113, 1115,1117, 1119, 1121, 1123, 1125, 1127, 1129, 1131, 1133, 1135, 1137, 1139,1141, 1143, 1145, 1147, 1149, 1151, 1153, 1155, 1157, 1159, 1161, 1163,1165, 1167, 1169, 1171, 1173, 1175, 1177, 1179, 1181, 1183, 1185, 1187,1189, 1191, 1193, 1195, 1197, 1199, 1201, 1203, 1205, 1207, 1209, 1211,1213, 1215, 1217, 1219, 1221, 1223, 1225, 1227, 1229, 1231, 1233, 1235,1237, 1239, 1241, 1243, 1245, 1247, 1249, 1251, 1253, 1255, 1257, 1259,1261, 1263, 1265, 1267, 1269, 1271, 1273, 1275, 1277, 1279, 1281, 1283,1285, 1287, 1289, 1291, 1293, 1295, 1297, 1299, 1301, 1303, 1305, 1307,1309, 1310, 1311, 1313, 1315, 1317, 1319, 1321, 1323, 1325, 1327, 1329,1331, 1333, 1335, 1337, 1339, 1341, 1343, 1345, 1347, 1349, 1351, 1353,1355, 1357, 1359, 1361, 1363, 1365, 1367, 1369, 1371, 1373, 1375, 1377,1379, 1381, 1383, 1385, 1387, 1389, 1391, 1393, 1395, 1397, 1399, 1401,1403, 1405, 1407, 1409, 1411, 1413, 1415, 1417, 1419, 1421, 1423, 1425,1427, 1429, 1431, 1433, 1435, 1437, 1439, 1441, 1443, 1445, 1447, 1449,1451, 1453, 1455, 1457, 1459, 1461, 1463, 1465, 1467, 1469, 1471, 1473,1475, 1477, 1479, 1481, 1483, 1485, 1487, 1489, 1491, 1493, 1495, 1497,1499, 1501, 1503, 1505, 1507, 1509, 1511, 1513, 1515, 1517, 1519, 1521,1523, 1525, 1527, 1529, 1531, 1533, 1535, 1537, 1539, 1541, 1543, 1545,1547, 1549, 1551, 1553, 1555, 1557, 1559, 1561, 1563, 1565, 1567, 1569,1571, 1573, 1575, 1577, 1579, 1581, 1583, 1585, 1587, 1589, 1591, 1593,1595, 1597, 1599, 1601, 1603, 1605, 1607, 1609, 1611, 1613, 1615, 1617,1619, 1621, 1623, 1625, 1627, 1629, 1631, 1633, 1635, 1637, 1639, 1641,1643, 1645, 1647, 1649, 1651, 1653, 1655, 1657, 1659, 1661, 1663, 1665,1667, 1669, 1671, 1673, 1675, 1677, 1679, 1681, 1683, 1685, 1687, 1689,1691, 1693, 1695, 1697, 1699, 1701, 1703, 1705, 1707, 1709, 1711, 1713,1715, 1717, 1719, 1721, 1723, 1725, 1727, 1729, 1731, 1733, 1735, 1737,1739, 1741, 1743, 1745, 1747, 1749, 1751, 1753, 1755, 1757, 1759, 1761,1763, 1765, 1767, 1769, 1771, 1773, 1775, 1777, 1779, 1781, 1783, 1785,1787, 1789, 1791, 1793, 1795, 1797, 1799, 1801, 1803, 1805, 1807, 1809,1811, 1813, 1815, 1817, 1819, 1821, 1823, 1825, 1827, 1829, 1831, 1833,1835, 1837, 1839, 1841, 1843, 1845, 1847, 1849, 1851, 1853, 1855, 1857,1859, 1861, 1863, 1865, 1867, 1869, 1871, 1873, 1875, 1877, 1879, 1881,1883, 1885, 1887, 1889, 1891, 1893, 1895, 1897, 1899, 1901, 1903, 1905,1907, 1909, 1911, 1913, 1915, 1917, 1919, 1921, 1923, 1925, 1927, 1929,1931, 1933, 1935, 1937, 1939, 1941, 1943, 1945, 1947, 1949, 1951, 1953,1955, 1957, 1959, 1961, 1963, 1965, 1967, 1969, 1971, 1973, 1975, 1977,1979, 1981, 1983, 1985, 1987, 1989, 1991, 1993, 1995, 1997, 1999, 2001,2003, 2005, 2007, 2009, 2011, 2013, 2015, 2017, 2019, 2021, 2023, 2025,2027, 2029, 2031, 2033, 2035, 2037, 2039, 2041, 2043, 2045, 2047, 2049,2051, 2053, 2055, 2057, 2059, 2061, 2063, 2065, 2067, 2069, 2071, 2073,2075, 2077, 2079, 2081, 2083, 2085, 2087, 2089, 2091, 2093, 2095, 2097,2099, 2101, 2103, 2105, 2107, 2109, 2111, 2113, 2115, 2117, 2119, 2121,2123, 2125, 2127, 2129, 2131, 2133, 2135, 2137, 2139, 2141, 2143, 2145,2147, 2149, 2151, 2153, 2155, 2157, 2159, 2161, 2163, 2165, 2167, 2169,2171, 2173, 2175, 2177, 2179, 2181, 2183, 2185, 2187, 2189, 2191, 2193,2195, 2197, 2199, 2201, 2203, 2205, 2207, 2209, 2211, 2213, 2215, 2217,2219, 2221, 2223, 2225, 2227, 2229, 2231, 2233, 2235, 2237, 2239, 2241,2243, 2245, 2247, 2249, 2251, 2253, 2255, 2257, 2259, 2261, 2263, 2265,2267, 2269, 2271, 2273, 2275, 2277, 2279, 2281, 2283, 2285, 2287, 2289,2291, 2293, 2295, 2297, 2299, 2301, 2303, 2305, 2307, 2309, 2311, 2313,2315, 2317, 2319, 2321, 2323, 2325, 2327, 2329, 2331, 2333, 2335, 2337,2339, 2341, 2343, 2345, 2347, 2349, 2351, 2353, 2355, 2357, 2359, 2361,2363, 2365, 2367, 2369, 2371, 2373, 2375, 2377, 2379, 2381, 2383, 2385,2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407, 2409,2411, 2413, 2415, 2417, 2419, 2421, 2423, 2425, 2427, 2429, 2431, 2433,2435, 2437, 2439, 2441, 2443, 2445, 2447, 2449, 2451, 2453, 2455, 2457,2459, 2461, 2463, 2465, 2467, 2469, 2471, 2473, 2475, 2477, 2479, 2481,2483, 2485, 2487, 2489, 2491, 2493, 2495, 2497, 2499, 2501, 2503, 2505,2507, 2509, 2511, 2513, 2515, 2517, 2519, 2521, 2523, 2525, 2527, 2529,2531, 2533, 2635, 2537, 2539, 2541, 2543, 2545, 2547, 2549, 2551, 2553,2555, 2557, 2559, 2561, 2563, 2565, 2567, 2569, 2571, 2573, 2575, 2577,2579, 2581, 2583, 2585, 2587, 2589, 2591, 2593, 2595, 2597, 2599, 2601,2603, 2605, 2607, 2609, 2611, 2613, 2615, 2617, 2619, 2621, 2623, 2625,2627, 2629, 2631, 2633, 2635, 2637, 2639, 2641, 2643, 2645, 2647, 2649,2651, 2653, 2655, 2657, 2659, 2661, 2663, 2665, 2667, 2669, 2671, 2673,2675, 2677, 2679, 2681, 2683, 2685, 2687, 2689, 2691, 2693, 2695, 2697,2699, 2701, 2703, 2705, 2707, 2709, 2711, 2713, 2715, 2717, 2719, 2721,2723, 2725, 2727, 2729, 2731, 2733, 2735, 2737, 2739, 2741, 2743, 2745,2747, 2749, 2751, 2753, 2755, 2757, 2759, 2761, 2763, 2765, 2767, 2769,2771, 2773, 2775, 2777, 2779, 2781, 2783, 2785, 2787, 2789, 2791, 2793,2795, 2797, 2799, 2801, 2803, 2805, 2807, 2809, 2811, 2813, 2815, 2817,2819, 2821, 2823, 2825, 2827, 2829, 2831, 2833, 2835, 2837, 2839, 2841,2843, 2845, and 2847, respectively.

The present invention also provides an isolated or recombinant HHDHpolypeptide having an amino acid sequence that has a substitution,deletion, and/or insertion of from one to twenty amino acid residues inany one of the following sequences: SEQ ID NO: 2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88,90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118,120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146,148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174,176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202,204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230,232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258,260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286,288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314,316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342,344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 368, 370, 372,374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400,402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428,430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456,458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484,486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512,514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540,542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568,570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596,598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624,626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652,654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680,682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708,710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736,738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764,766, 768, 770, 772, 774, 776, 778, 780, 782, 784, 786, 788, 790, 792,794, 796, 798, 800, 802, 804, 806, 808, 810, 812, 814, 816, 818, 820,822, 824, 826, 828, 830, 832, 834, 836, 838, 840, 842, 844, 846, 848,850, 852, 854, 856, 858, 860, 862, 864, 866, 868, 870, 872, 874, 876,878, 880, 882, 884, 886, 888, 890, 892, 894, 896, 898, 900, 902, 904,906, 908, 910, 912, 914, 916, 918, 920, 922, 924, 926, 928, 930, 932,934, 936, 938, 940, 942, 944, 946, 948, 950, 952, 954, 956, 958, 960,962, 964, 966, 968, 970, 972, 974, 976, 978, 980, 982, 984, 986, 988,990, 992, 994, 996, 998, 1000, 1002, 1004, 1006, 1008, 1010, 1012, 1014,1016, 1018, 1020, 1022, 1024, 1026, 1028, 1030, 1032, 1034, 1036, 1038,1040, 1042, 1044, 1046, 1048, 1050, 1052, 1054, 1056, 1058, 1060, 1062,1064, 1066, 1068, 1070, 1072, 1074, 1076, 1078, 1080, 1082, 1084, 1086,1088, 1090, 1092, 1094, 1096, 1098, 1100, 1102, 1104, 1106, 1108, 1110,1112, 1114, 1116, 1118, 1120, 1122, 1124, 1126, 1128, 1130, 1132, 1134,1136, 1138, 1140, 1142, 1144, 1146, 1148, 1150, 1152, 1154, 1156, 1158,1160, 1162, 1164, 1166, 1168, 1170, 1172, 1174, 1176, 1178, 1180, 1182,1184, 1186, 1188, 1190, 1192, 1194, 1196, 1198, 1200, 1202, 1204, 1206,1208, 1210, 1212, 1214, 1216, 1218, 1220, 1222, 1224, 1226, 1228, 1230,1232, 1234, 1236, 1238, 1240, 1242, 1244, 1246, 1248, 1250, 1252, 1254,1256, 1258, 1260, 1262, 1264, 1266, 1268, 1270, 1272, 1274, 1276, 1278,1280, 1282, 1284, 1286, 1288, 1290, 1292, 1294, 1296, 1298, 1300, 1302,1304, 1306, 1308, 1310, 1312, 1314, 1316, 1318, 1320, 1322, 1324, 1326,1328, 1330, 1332, 1334, 1336, 1338, 1340, 1342, 1344, 1346, 1348, 1350,1352, 1354, 1356, 1358, 1360, 1362, 1364, 1366, 1368, 1370, 1372, 1374,1376, 1378, 1380, 1382, 1384, 1386, 1388, 1390, 1392, 1394, 1396, 1398,1400, 1402, 1404, 1406, 1408, 1410, 1412, 1414, 1416, 1418, 1420, 1422,1424, 1426, 1428, 1430, 1432, 1434, 1436, 1438, 1440, 1442, 1444, 1446,1448, 1450, 1452, 1454, 1456, 1458, 1460, 1462, 1464, 1466, 1468, 1470,1472, 1474, 1476, 1478, 1480, 1482, 1484, 1486, 1488, 1490, 1492, 1494,1496, 1498, 1500, 1502, 1504, 1506, 1508, 1510, 1512, 1514, 1516, 1518,1520, 1522, 1524, 1526, 1528, 1530, 1532, 1534, 1536, 1538, 1540, 1542,1544, 1546, 1548, 1550, 1552, 1554, 1556, 1558, 1560, 1562, 1564, 1566,1568, 1570, 1572, 1574, 1576, 1578, 1580, 1582, 1584, 1586, 1588, 1590,1592, 1594, 1596, 1598, 1600, 1602, 1604, 1606, 1608, 1610, 1612, 1614,1616, 1618, 1620, 1622, 1624, 1626, 1628, 1630, 1632, 1634, 1636, 1638,1640, 1642, 1644, 1646, 1648, 1650, 1652, 1654, 1656, 1658, 1660, 1662,1664, 1666, 1668, 1670, 1672, 1674, 1676, 1678, 1680, 1682, 1684, 1686,1688, 1690, 1692, 1694, 1696, 1698, 1700, 1702, 1704, 1706, 1708, 1710,1720, 1722, 1724, 1726, 1728, 1730, 1732, 1734, 1736, 1738, 1740, 1742,1744, 1746, 1748, 1750, 1752, 1754, 1756, 1758, 1760, 1762, 1764, 1766,1768, 1770, 1772, 1774, 1776, 1778, 1780, 1782, 1784, 1786, 1788, 1790,1792, 1794, 1796, 1798, 1800, 1802, 1804, 1806, 1808, 1810, 1812, 1814,1816, 1818, 1820, 1822, 1824, 1826, 1828, 1830, 1832, 1834, 1836, 1838,1840, 1842, 1844, 1846, 1848, 1850, 1852, 1854, 1856, 1858, 1860, 1862,1864, 1866, 1868, 1870, 1872, 1874, 1876, 1878, 1880, 1882, 1884, 1886,1888, 1890, 1892, 1894, 1896, 1898, 1900, 1902, 1904, 1906, 1908, 1910,1912, 1914, 1916, 1918, 1920, 1922, 1924, 1926, 1928, 1930, 1932, 1934,1936, 1938, 1940, 1942, 1944, 1946, 1948, 1950, 1952, 1954, 1956, 1958,1960, 1962, 1964, 1966, 1968, 1970, 1972, 1974, 1976, 1978, 1980, 1982,1984, 1986, 1988, 1990, 1992, 1994, 1996, 1998, 2000, 2002, 2004, 2006,2008, 2010, 2012, 2014, 2016, 2018, 2020, 2022, 2024, 2026, 2028, 2030,2032, 2034, 2036, 2038, 2040, 2042, 2044, 2046, 2048, 2050, 2052, 2054,2056, 2058, 2060, 2062, 2064, 2066, 2068, 2070, 2072, 2074, 2076, 2078,2080, 2082, 2084, 2086, 2088, 2090, 2092, 2094, 2096, 2098, 2100, 2102,2104, 2106, 2108, 2110, 2112, 2114, 2116, 2118, 2120, 2122, 2124, 2126,2128, 2130, 2132, 2134, 2136, 2138, 2140, 2142, 2144, 2146, 2148, 2150,2152, 2154, 2156, 2158, 2160, 2162, 2164, 2166, 2168, 2170, 2172, 2174,2176, 2178, 2180, 2182, 2184, 2186, 2188, 2190, 2192, 2194, 2196, 2198,2200, 2202, 2204, 2206, 2208, 2210, 2212, 2214, 2216, 2218, 2220, 2222,2224, 2226, 2228, 2230, 2232, 2234, 2236, 2238, 2240, 2242, 2244, 2246,2248, 2250, 2252, 2254, 2256, 2258, 2260, 2262, 2264, 2266, 2268, 2270,2272, 2274, 2276, 2278, 2280, 2282, 2284, 2286, 2288, 2300, 2302, 2304,2306, 2308, 2310, 2312, 2314, 2316, 2318, 2320, 2322, 2324, 2326, 2328,2330, 2332, 2334, 2336, 2338, 2340, 2342, 2344, 2346, 2348, 2350, 2352,2354, 2356, 2358, 2360, 2362, 2364, 2366, 2368, 2370, 2372, 2374, 2376,2378, 2380, 2382, 2384, 2386, 2388, 2390, 2392, 2394, 2396, 2398, 2400,2402, 2404, 2406, 2408, 2410, 2412, 2414, 2416, 2418, 2420, 2422, 2424,2426, 2428, 2430, 2432, 2434, 2436, 2438, 2440, 2442, 2444, 2446, 2448,2450, 2452, 2454, 2456, 2458, 2460, 2462, 2464, 2466, 2468, 2470, 2472,2474, 2476, 2478, 2480, 2482, 2484, 2486, 2488, 2490, 2492, 2494, 2496,2498, 2500, 2502, 2504, 2506, 2508, 2510, 2512, 2514, 2516, 2518, 2520,2522, 2524, 2526, 2528, 2530, 2532, 2534, 2536, 2538, 2540, 2542, 2544,2546, 2548, 2550, 2552, 2554, 2556, 2558, 2560, 2562, 2564, 2566, 2568,2570, 2572, 2574, 2576, 2578, 2580, 2582, 2584, 2586, 2588, 2590, 2592,2594, 2596, 2598, 2600, 2602, 2604, 2606, 2608, 2610, 2612, 2614, 2616,2618, 2620, 2622, 2624, 2626, 2628, 2630, 2632, 2634, 2636, 2638, 2640,2642, 2644, 2646, 2648, 2650, 2652, 2654, 2656, 2658, 2660, 2662, 2664,2666, 2668, 2670, 2672, 2674, 2676, 2678, 2680, 2682, 2684, 2686, 2688,2690, 2700, 2702, 2704, 2706, 2708, 2710, 2712, 2714, 2716, 2718, 2720,2722, 2724, 2726, 2728, 2730, 2732, 2734, 2736, 2738, 2740, 2742, 2744,2746, 2748, 2750, 2752, 2754, 2756, 2758, 2760, 2762, 2764, 2766, 2768,2770, 2772, 2774, 2776, 2778, 2780, 2782, 2784, 2786, 2788, 2790, 2792,2794, 2796, 2798, 2800, 2802, 2804, 2806, 2808, 2810, 2812, 2814, 2816,2818, 2820, 2822, 2824, 2826, 2828, 2830, 2832, 2834, 2836, 2838, 2840,2842, 2844, 2846, or 2848. These HHDH polypeptides may have asubstitution, deletion, and/or insertion of from one to two, or from oneor two, to three, four, five, six, seven, eight, nine, ten, eleven,twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen,nineteen or twenty residues.

Typically, the HHDH polypeptides of paragraph 0135 have HHDH activitythat is at least 1.4 fold greater than the HHDH polypeptide of SEQ IDNO: 2, as measured in the assay of Example 5A; or (b) at least 1.5-foldgreater than the HHDH polypeptide of SEQ ID NO: 730, as measured in theassay of Example D.

Variants of the HHDH polypeptides of the present invention may begenerated using methods that are well known to those having ordinaryskill in the art. Libraries of these variants may be generated andscreened using the high throughput screen for presence of HHDH activitydescribed in Example 4A. In some instances it may be desirable toidentify halohydrin dehalogenases that exhibit activity in the presenceof cyanohydrin product inhibitor, e.g., ethyl(R)-4-cyano-3-hydroxybutyrate. A high throughput screen for identifyingsuch enzymes is provided in Example 4B. Variants that exhibit HHDHactivity in the presence of product cyanohydrin may be furthercharacterized using the assay described in Example 5B. It may bedesirable to identify HHDH polypeptides of the present invention thathave resistance to ECAA inhibition. A method of screening for resistanceto ECAA inhibition is provided in Example 5C. As described supra, amethod for assaying for the time to fully complete ECHB to HNHHDH-catalyzed conversion in a very low throughput, scaled up reactionis provided in Example 5D.

Methods for generating variant libraries are well known in the art. Forexample, mutagenesis and directed evolution methods can be readilyapplied to polynucleotides (such as, for example, wild-type HHDHencoding polynucleotides or the polynucleotides of the presentinvention) to generate variant libraries that can be expressed,screened, and assayed using the methods described herein. Mutagenesisand directed evolution methods are well known in the art. See, e.g.,Ling, et al., “Approaches to DNA mutagenesis: an overview,” Anal.Biochem., 254(2): 157-78 (1997); Dale, et al., “Oligonucleotide-directedrandom mutagenesis using the phosphorothioate method,” Methods Mol.Biol., 57:369-74 (1996); Smith, “In vitro mutagenesis,” Ann. Rev.Genet., 19:423-462 (1985); Botstein, et al., “Strategies andapplications of in vitro mutagenesis,” Science 229:1193-1201 (1985);Carter, “Site-directed mutagenesis,” Biochem. J., 237:1-7 (1986);Kramer, et al., “Point Mismatch Repair,” Cell 38:879-887 (1984); Wells,et al., “Cassette mutagenesis: an efficient method for generation ofmultiple mutations at defined sites,” Gene, 34:315-323 (1985); Minshull,et al., “Protein evolution by molecular breeding,” Current Opinion inChemical Biology, 3:284-290 (1999); Christians, et al., “Directedevolution of thymidine kinase for AZT phosphorylation using DNA familyshuffling,” Nature Biotechnology, 17:259-264 (1999); Crameri, et al.,“DNA shuffling of a family of genes from diverse species acceleratesdirected evolution,” Nature, 391:288-291; Crameri, et al., “Molecularevolution of an arsenate detoxification pathway by DNA shuffling,”Nature Biotechnology, 15:436-438 (1997); Zhang, et al., “Directedevolution of an effective fucosidase from a galactosidase by DNAshuffling and screening,” Proceedings of the National Academy ofSciencess, U.S.A., 94:45-4-4509; Crameri, et al., “Improved greenfluorescent protein by molecular evolution using DNA shuffling,” NatureBiotechnology, 14:315-319 (1996); Stemmer, “Rapid evolution of a proteinin vitro by DNA shuffling,” Nature, 370:389-391 (1994); Stemmer, “DNAshuffling by random fragmentation and reassembly: In vitro recombinationfor molecular evolution,” Proceedings of the National Academy ofSciences, U.S.A., 91:10747-10751 (1994); WO 95/22625; WO 97/0078; WO97/35966; WO 98/27230; WO 00/42651; and WO 01/75767.

In another embodiment, the present invention also provides a fragment ofthe HHDH polypeptides described herein having HHDH activity as detectedin the assay of Example 5A. These fragments are referred to herein as“HHDH fragments”. As used herein, the term “fragment” refers to apolypeptide having a deletion of from 1 to 15 amino acid residues fromthe carboxy terminus, the amino terminus, or both. In certainembodiments, the deletion is from 1 to 14 residues from the carboxyterminus, the amino terminus, or both. In some embodiments, the deletionmay be from 1 to 10 residues, or 1 to 5 residues from the carboxyterminus, the amino terminus, or both. HHDH fragments of the presentinvention include those that have at least 1.4 fold greater HHDHactivity than the activity of Agrobacterium sp. (wild-type) HHDH (SEQ IDNO: 2) in the assay of Example 5A; or at least 1.5-fold greater HHDHactivity than the activity of the HHDH polypeptide of SEQ ID NO: 730 inthe assay of Example 5D.

HHDH polypeptides of the present invention include isolated andrecombinant HHDH polypeptides having enhanced thermostability ascompared to the wildtype HHDH of SEQ ID NO: 2. Enhanced thermostabilitycan be readily determined by comparing the HHDH activity of the HHDHpolypeptide to wildtype HHDH (SEQ ID NO:2) in the medium throughputassay of Example 5B, which includes a two hour incubation at 50° C. HHDHpolypeptides having enhanced thermostability include those having aHistidine residue at position 72, wherein amino acid position isdetermined by optimal alignment of the amino acid sequence of the HHDHpolypeptide with SEQ ID NO: 2 (i.e., a Q72H mutation).

Each of the residue changes to an HHDH polypeptide was evaluated todetermine what relationship, if any, existed between the sequence changeand the desired function (e.g., increased HHDH enzymatic activity). Todo so, the sequence changes and resulting HHDH activity in members of alibrary generated by the method described in WO 00/42561 and mutagenesismethods described hereinbelow, were evaluated using the method disclosedin U.S. Ser. No. 10/379, 378 filed Mar. 3, 2003, entitled “Methods,systems, and software for identifying functional biomolecules” andincorporated herein by reference. See also, R. Fox et al., J. Theor.Biol. 234 (2005) and R. Fox et al., Protein Eng. 16 (2003), both ofwhich are incorporated herein by reference. Based upon this method,codons encoding important residues at certain positions that appear tocorrelate favorably to activity were identified and incorporated intothe polynucleotides of a subsequently generated combinatorial library.In other words, the polynucleotides encoding the desired change weregenerated, expressed and then screened. The method is again applied tothe resulting sequences and the HHDH activity is characterized. Theresults are again utilized to select those residue changes that enhanceHHDH activity for programming into the next library. Using this method,the functionality of various sequence changes is subject to immediateevaluation. The residue changes at various residue positions thatprovide for enhanced enzymatic activity relative to the wild-type HHDHare disclosed herein in the sequences and elsewhere as preferredresidues at identified positions. Therefore, the above directedevolution scheme, which utilizes the high throughput screen of Example 4and the assay of Example 5A, can be used to identify further variants ofthe HHDH polypeptides described herein. Further characterization of thevariant HHDH polypeptides can be conducted using the assays of Example5B, 5C, or 5D, which are described in more detail above.

Sequence-activity analyses indicated that the above-described mutationsappeared favorable with respect to increasing HHDH activity relative tothe wildtype HHDH of SEQ ID NO: 2. Sequence-activity analysis wasperformed in accordance with the methods described in WO 03/075129, U.S.Ser. No. 10/379, 378 filed Mar. 3, 2003, and R. Fox et al., “Optimizingthe search algorithm for protein engineering by directed evolution,”Protein Eng. 16(8):589-597 (2003), both of which are incorporated hereinby reference. See also R. Fox et al., “Directed molecular evolution bymachine learning and the influence of nonlinear interactions,” J. Theor.Biol. 234(2):187-199 (2005), which is incorporated herein by reference.This analysis also indicated that the Asparagine residue at position 79appeared beneficial with respect to HHDH activity in connection with theHHDH polypeptides described herein. Thus, the present invention providesan isolated or recombinant HHDH polypeptide having the features of anyone of the HHDH polypeptide embodiments described herein, wherein theamino acid sequence of the HHDH polypeptide further comprises anAsparagine residue at position 79. Similarly, Proline at position 175and Tyrosine at position 187 appeared to be favorable residues at thosepositions. Therefore, HHDH polypeptides of the present invention includean isolated and recombinant HHDH polypeptide of any one of theembodiments described herein, further comprising an amino acid residueselected from the group consisting of Proline at position 175 andTyrosine at position 187.

Structural analysis of the mutations described herein revealed that theyare distributed throughout the protein, with some of the mutations inthe active site within 7.5 Å of the bound product in the HheC structure.See R. M. DeJong et al., EMBO Journal 22:4933 (2003) for the tertiarystructure of HHDH, which is incorporated herein by reference. Of theeleven residues that directly interact with the product analog orCl⁻ion, four are mutated: F186Y, T134A, Y177A/G, and L178C/M/V (relativeto the wildtype HHDH of SEQ ID NO: 2). Of the contacting positionswithout mutations in this set, two are part of the catalytic triad ofthe HHDH polypeptide: S132 and Y145. Accordingly, the present inventionprovides an isolated or recombinant HHDH polypeptide having the featuresof any one of the HHDH polypeptide embodiments described herein, whereinthe HHDH polypeptide further comprises a Serine at position 132 and/or aTyrosine at position 145. Structural analysis revealed that tworesidues, W139 and N176, were mutated into an apparent salt bridge intwo intermediate active variants: W139R/N176E and W139D/N176R (withreference to SEQ ID NO: 2). Both of these proteins exhibited greaterHHDH activity relative to wildtype HHDH (SEQ ID NO: 2). Residue F12 inthe wildtype HHDH (SEQ ID NO: 2), which replaces a glycine from theGX₃GXG motif of the evolutionarily related SDR superfamily (see DeJonget al., Id.), could be replaced by I, L or Y with neutral effects.

The present invention includes conservatively modified variants of theHHDH polypeptides described herein. These variants have conservativesubstitutions made in their amino acid sequences. Examples ofconservative substitutions are within the group of basic amino acids(arginine, lysine and histidine), acidic amino acids (glutamic acid andaspartic acid), polar amino acids (glutamine and asparagines),hydrophobic amino acids (leucine, isoleucine and valine), aromatic aminoacids (phenylalanine, tryptophan and tyrosine), and small amino acids(glycine, alanine, serine, threonine, proline, cysteine and methionine).Amino acid substitutions which do not generally alter the specificactivity are known in the art and are described, for example, by H.Neurath and R. L. Hill, 1979, in “The Proteins,” Academic Press, NewYork. The most commonly occurring exchanges are Ala/Ser, Val/Ile,Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe,Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly aswell as these in reverse.

Conservatively substituted variations of the HHDH polypeptides of thepresent invention include substitutions of a small percentage, typicallyless than 5%, more typically less than 2%, and often less than 1% of theamino acids of the polypeptide sequence, with a conservatively selectedamino acid of the same conservative substitution group. The addition ofsequences which do not alter the encoded activity of an HHDHpolynucleotide, such as the addition of a non-functional or non-codingsequence, is considered a conservative variation of the HHDHpolynucleotide.

HHDH Polynucleotides

The present invention provides isolated or recombinant polynucleotidesthat encode any of the above-described HHDH polypeptides.

Exemplary HHDH polynucleotides include those corresponding to SEQ ID NO:3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 33, 35, 37, 39, 41,43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77,79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137,139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165,167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193,195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221,223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249,251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277,279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305,307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333,335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361,363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389,391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417,419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445,447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 4712, 473,475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501,503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529,531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557,559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585,587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613,615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641,643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669,671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697,699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725,727, 729, 731, 733, 735, 739, 741, 743, 745, 747, 749, 751, 753, 755,757, 759, 761, 763, 765, 767, 769, 771, 773, 775, 777, 779, 781, 783,785, 787, 789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 809, 811,813, 815, 817, 819, 821, 823, 825, 827, 829, 831, 833, 835, 837, 839,841, 843, 845, 847, 849, 851, 853, 855, 857, 859, 861, 863, 865, 867,869, 871, 873, 875, 877, 879, 881, 883, 885, 887, 889, 891, 893, 895,897, 899, 901, 903, 905, 907, 909, 911, 913, 915, 917, 919, 921, 923,925, 927, 929, 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951,953, 955, 957, 959, 961, 963, 965, 967, 969, 971, 973, 975, 977, 979,981, 983, 985, 987, 989, 991, 993, 995, 997, 999, 1001, 1103, 1005,1007, 1009, 1011, 1013, 1015, 1017, 1019, 1021, 1023, 1025, 1027, 1029,1031, 1033, 1035, 1037, 1039, 1041, 1043, 1045, 1047, 1049, 1051, 1053,1055, 1057, 1059, 1061, 1063, 1065, 1067, 1069, 1071, 1073, 1075, 1077,1079, 1081, 1083, 1085, 1087, 1089, 1091, 1093, 1095, 1097, 1099, 1101,1103, 1105, 1107, 1109, 1111, 1113, 1115, 1117, 1119, 1121, 1123, 1125,1127, 1129, 1131, 1133, 1135, 1137, 1139, 1141, 1143, 1145, 1147, 1149,1151, 1153, 1155, 1157, 1159, 1161, 1163, 1165, 1167, 1169, 1171, 1173,1175, 1177, 1179, 1181, 1183, 1185, 1187, 1189, 1191, 1193, 1195, 1197,1199, 1201, 1203, 1205, 1207, 1209, 1211, 1213, 1215, 1217, 1219, 1221,1223, 1225, 1227, 1229, 1231, 1233, 1235, 1237, 1239, 1241, 1243, 1245,1247, 1249, 1251, 1253, 1255, 1257, 1259, 1261, 1263, 1265, 1267, 1269,1271, 1273, 1275, 1277, 1279, 1281, 1283, 1285, 1287, 1289, 1291, 1293,1295, 1297, 1299, 1301, 1303, 1305, 1307, 1309, 1310, 1311, 1313, 1315,1317, 1319, 1321, 1323, 1325, 1327, 1329, 1331, 1333, 1335, 1337, 1339,1341, 1343, 1345, 1347, 1349, 1351, 1353, 1355, 1357, 1359, 1361, 1363,1365, 1367, 1369, 1371, 1373, 1375, 1377, 1379, 1381, 1383, 1385, 1387,1389, 1391, 1393, 1395, 1397, 1399, 1401, 1403, 1405, 1407, 1409, 1411,1413, 1415, 1417, 1419, 1421, 1423, 1425, 1427, 1429, 1431, 1433, 1435,1437, 1439, 1441, 1443, 1445, 1447, 1449, 1451, 1453, 1455, 1457, 1459,1461, 1463, 1465, 1467, 1469, 1471, 1473, 1475, 1477, 1479, 1481, 1483,1485, 1487, 1489, 1491, 1493, 1495, 1497, 1499, 1501, 1503, 1505, 1507,1509, 1511, 1513, 1515, 1517, 1519, 1521, 1523, 1525, 1527, 1529, 1531,1533, 1535, 1537, 1539, 1541, 1543, 1545, 1547, 1549, 1551, 1553, 1555,1557, 1559, 1561, 1563, 1565, 1567, 1569, 1571, 1573, 1575, 1577, 1579,1581, 1583, 1585, 1587, 1589, 1591, 1593, 1595, 1597, 1599, 1601, 1603,1605, 1607, 1609, 1611, 1613, 1615, 1617, 1619, 1621, 1623, 1625, 1627,1629, 1631, 1633, 1635, 1637, 1639, 1641, 1643, 1645, 1647, 1649, 1651,1653, 1655, 1657, 1659, 1661, 1663, 1665, 1667, 1669, 1671, 1673, 1675,1677, 1679, 1681, 1683, 1685, 1687, 1689, 1691, 1693, 1695, 1697, 1699,1701, 1703, 1705, 1707, 1709, 1711, 1713, 1715, 1717, 1719, 1721, 1723,1725, 1727, 1729, 1731, 1733, 1735, 1737, 1739, 1741, 1743, 1745, 1747,1749, 1751, 1753, 1755, 1757, 1759, 1761, 1763, 1765, 1767, 1769, 1771,1773, 1775, 1777, 1779, 1781, 1783, 1785, 1787, 1789, 1791, 1793, 1795,1797, 1799, 1801, 1803, 1805, 1807, 1809, 1811, 1813, 1815, 1817, 1819,1821, 1823, 1825, 1827, 1829, 1831, 1833, 1835, 1837, 1839, 1841, 1843,1845, 1847, 1849, 1851, 1853, 1855, 1857, 1859, 1861, 1863, 1865, 1867,1869, 1871, 1873, 1875, 1877, 1879, 1881, 1883, 1885, 1887, 1889, 1891,1893, 1895, 1897, 1899, 1901, 1903, 1905, 1907, 1909, 1911, 1913, 1915,1917, 1919, 1921, 1923, 1925, 1927, 1929, 1931, 1933, 1935, 1937, 1939,1941, 1943, 1945, 1947, 1949, 1951, 1953, 1955, 1957, 1959, 1961, 1963,1965, 1967, 1969, 1971, 1973, 1975, 1977, 1979, 1981, 1983, 1985, 1987,1989, 1991, 1993, 1995, 1997, 1999, 2001, 2003, 2005, 2007, 2009, 2011,2013, 2015, 2017, 2019, 2021, 2023, 2025, 2027, 2029, 2031, 2033, 2035,2037, 2039, 2041, 2043, 2045, 2047, 2049, 2051, 2053, 2055, 2057, 2059,2061, 2063, 2065, 2067, 2069, 2071, 2073, 2075, 2077, 2079, 2081, 2083,2085, 2087, 2089, 2091, 2093, 2095, 2097, 2099, 2101, 2103, 2105, 2107,2109, 2111, 2113, 2115, 2117, 2119, 2121, 2123, 2125, 2127, 2129, 2131,2133, 2135, 2137, 2139, 2141, 2143, 2145, 2147, 2149, 2151, 2153, 2155,2157, 2159, 2161, 2163, 2165, 2167, 2169, 2171, 2173, 2175, 2177, 2179,2181, 2183, 2185, 2187, 2189, 2191, 2193, 2195, 2197, 2199, 2201, 2203,2205, 2207, 2209, 2211, 2213, 2215, 2217, 2219, 2221, 2223, 2225, 2227,2229, 2231, 2233, 2235, 2237, 2239, 2241, 2243, 2245, 2247, 2249, 2251,2253, 2255, 2257, 2259, 2261, 2263, 2265, 2267, 2269, 2271, 2273, 2275,2277, 2279, 2281, 2283, 2285, 2287, 2289, 2291, 2293, 2295, 2297, 2299,2301, 2303, 2305, 2307, 2309, 2311, 2313, 2315, 2317, 2319, 2321, 2323,2325, 2327, 2329, 2331, 2333, 2335, 2337, 2339, 2341, 2343, 2345, 2347,2349, 2351, 2353, 2355, 2357, 2359, 2361, 2363, 2365, 2367, 2369, 2371,2373, 2375, 2377, 2379, 2381, 2383, 2385, 2387, 2389, 2391, 2393, 2395,2397, 2399, 2401, 2403, 2405, 2407, 2409, 2411, 2413, 2415, 2417, 2419,2421, 2423, 2425, 2427, 2429, 2431, 2433, 2435, 2437, 2439, 2441, 2443,2445, 2447, 2449, 2451, 2453, 2455, 2457, 2459, 2461, 2463, 2465, 2467,2469, 2471, 2473, 2475, 2477, 2479, 2481, 2483, 2485, 2487, 2489, 2491,2493, 2495, 2497, 2499, 2501, 2503, 2505, 2507, 2509, 2511, 2513, 2515,2517, 2519, 2521, 2523, 2525, 2527, 2529, 2531, 2533, 2635, 2537, 2539,2541, 2543, 2545, 2547, 2549, 2551, 2553, 2555, 2557, 2559, 2561, 2563,2565, 2567, 2569, 2571, 2573, 2575, 2577, 2579, 2581, 2583, 2585, 2587,2589, 2591, 2593, 2595, 2597, 2599, 2601, 2603, 2605, 2607, 2609, 2611,2613, 2615, 2617, 2619, 2621, 2623, 2625, 2627, 2629, 2631, 2633, 2635,2637, 2639, 2641, 2643, 2645, 2647, 2649, 2651, 2653, 2655, 2657, 2659,2661, 2663, 2665, 2667, 2669, 2671, 2673, 2675, 2677, 2679, 2681, 2683,2685, 2687, 2689, 2691, 2693, 2695, 2697, 2699, 2701, 2703, 2705, 2707,2709, 2711, 2713, 2715, 2717, 2719, 2721, 2723, 2725, 2727, 2729, 2731,2733, 2735, 2737, 2739, 2741, 2743, 2745, 2747, 2749, 2751, 2753, 2755,2757, 2759, 2761, 2763, 2765, 2767, 2769, 2771, 2773, 2775, 2777, 2779,2781, 2783, 2785, 2787, 2789, 2791, 2793, 2795, 2797, 2799, 2801, 2803,2805, 2807, 2809, 2811, 2813, 2815, 2817, 2819, 2821, 2823, 2825, 2827,2829, 2831, 2833, 2835, 2837, 2839, 2841, 2843, 2845, 2847, andcomplementary sequences thereof.

Those having ordinary skill in the art will readily appreciate that dueto the degeneracy of the genetic code, a multitude of nucleotidesequences encoding HHDH polypeptides of the present invention exist.Table I is a Codon Table that provides the synonymous codons for eachamino acid. For example, the codons AGA, AGG, CGA, CGC, CGG, and CGU allencode the amino acid arginine. Thus, at every position in the nucleicacids of the invention where an arginine is specified by a codon, thecodon can be altered to any of the corresponding codons described abovewithout altering the encoded polypeptide. It is understood that U in anRNA sequence corresponds to T in a DNA sequence.

TABLE 1 Codon Table Amino acids Codon Alanine Ala A GGA GCC GCG GCUCysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu EGAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGUHistidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAAAAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUGAsparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln QCAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCAUCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUUTryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

Such “silent variations” are one species of “conservative” variation.One of ordinary skill in the art will recognize that each codon in anucleic acid (except AUG, which is ordinarily the only codon formethionine) can be modified by standard techniques to encode afunctionally identical polypeptide. Accordingly, each silent variationof a nucleic acid which encodes a polypeptide is implicit in anydescribed sequence. The invention contemplates and provides each andevery possible variation of nucleic acid sequence encoding a polypeptideof the invention that could be made by selecting combinations based onpossible codon choices. These combinations are made in accordance withthe standard triplet genetic code (set forth in Table 1), as applied tothe polynucleotide sequences of the present invention.

A group of two or more different codons that, when translated in thesame context, all encode the same amino acid, are referred to herein as“synonymous codons.” HHDH polynucleotides of the present invention maybe codon optimized for expression in a particular host organism bymodifying the polynucleotides to conform with the optimum codon usage ofthe desired host organism. Those having ordinary skill in the art willrecognize that tables and other references providing preferenceinformation for a wide range of organisms are readily available Seee.g., Henaut and Danchin in “Escherichia coli and Salmonella,”Neidhardt, et al. Eds., ASM Pres, Washington D.C. (1996), pp. 2047-2066.

An exemplary HHDH variant polynucleotide sequence of the presentinvention is provided as SEQ ID NO: 31, which expresses well in E. coli.This polynucleotide is a variant of SEQ ID NO: 1 that expresses thepolypeptide corresponding to SEQ ID NO: 2 from E. coli at a level ofabout 4½ fold higher than the amount expressed from SEQ ID NO: 1 (i.e.,HHDH-encoding polynucleotide encoding native HHDH from Agrobacteriumsp.).

In some embodiments of the present invention, certain codons arepreferred when the following residues are employed in the HHDHpolypeptides of the present invention: ATT encoding Isoleucine at aminoacid position 5; AAG encoding Lysine at amino acid position 36; ATTencoding Isoleucine at amino acid position 63; GAG encoding Glutamicacid at amino acid position 95; and CCC encoding Proline at amino acidposition 188. The amino acid position referred to above is thecorresponding amino acid position in SEQ ID NO: 2, when the inventionHHDH polypeptides are aligned with SEQ ID NO: 2.

The terms “conservatively modified variations” and “conservativevariations” are used interchangeably herein to refer to those nucleicacids that encode identical or essentially identical amino acidsequences, or in the situation where the nucleic acids are not codingsequences, the term refers to nucleic acids that are identical. One ofordinary skill in the art will recognize that individual substitutions,deletions or additions which alter, add or delete a single amino acid ora small percentage of amino acids in an encoded sequence are consideredconservatively modified variations where the alterations result in oneor more of the following: the deletion of an amino acid, addition of anamino acid, or substitution of an amino acid with a chemically similaramino acid. When more than one amino acid is affected, the percentage istypically less than 5% of amino acid residues over the length of theencoded sequence, and more typically less than 2%. References providingamino acids that are considered conservative substitutions for oneanother are well known in the art.

Polynucleotides of the present invention can be prepared using methodsthat are well known in the art. Typically, oligonucleotides of up toabout 120 bases are individually synthesized, then joined (e.g., byenzymatic or chemical ligation methods, or polymerase-mediated methods)to form essentially any desired continuous sequence. For example,polynucleotides of the present invention can be prepared by chemicalsynthesis using, e.g., the classical phosphoramidite method described byBeaucage, et al. (1981) Tetrahedron Letters, 22:1859-69, or the methoddescribed by Matthes, et al. (1984) EMBO J., 3:801-05, e.g., as istypically practiced in automated synthetic methods. According to thephosphoramidite method, oligonucleotides are synthesized, e.g., in anautomatic DNA synthesizer, purified, annealed, ligated and cloned inappropriate vectors.

In addition, essentially any nucleic acid can be custom ordered from anyof a variety of commercial sources, such as The Midland CertifiedReagent Company (Midland, Tex.), The Great American Gene Company(Ramona, Calif.), ExpressGen Inc. (Chicago, Ill.), Operon TechnologiesInc. (Alameda, Calif.), and many others.

Polynucleotides may also be synthesized by well-known techniques asdescribed in the technical literature. See, e.g., Carruthers, et al.,Cold Spring Harbor Symp. Quant. Biol., 47:411-418 (1982) and Adams, etal., J. Am. Chem. Soc., 105:661 (1983). Double stranded DNA fragmentsmay then be obtained either by synthesizing the complementary strand andannealing the strands together under appropriate conditions, or byadding the complementary strand using DNA polymerase with an appropriateprimer sequence.

General texts which describe molecular biological techniques usefulherein, including the use of vectors, promoters and many other relevanttopics, include Berger and Kimmel, Guide to Molecular CloningTechniques, Methods in Enzymology volume 152 Academic Press, Inc., SanDiego, Calif. (Berger); Sambrook et al., Molecular Cloning—A LaboratoryManual (2nd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1989 (“Sambrook”) and Current Protocols in MolecularBiology, F. M. Ausubel et al., eds., Current Protocols, a joint venturebetween Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.,(supplemented through 1999) (“Ausubel”). Examples of protocolssufficient to direct persons of skill through in vitro amplificationmethods, including the polymerase chain reaction (PCR) the ligase chainreaction (LCR), Qβ-replicase amplification and other RNA polymerasemediated techniques (e.g., NASBA), e.g., for the production of thehomologous nucleic acids of the invention are found in Berger, Sambrook,and Ausubel, as well as Mullis et al., (1987) U.S. Pat. No. 4,683,202;PCR Protocols A Guide to Methods and Applications (Innis et al. eds)Academic Press Inc. San Diego, Calif. (1990) (Innis); Arnheim & Levinson(Oct. 1, 1990) C&EN 3647; The Journal Of NIH Research (1991) 3, 81-94;(Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86, 1173; Guatelli et al.(1990) Proc. Natl. Acad. Sci. USA 87, 1874; Lomell et al. (1989) J.Clin. Chem 35, 1826; Landegren et al., (1988) Science 241, 1077-1080;Van Brunt (1990) Biotechnology 8, 291-294; Wu and Wallace, (1989) Gene4, 560; Barringer et al. (1990) Gene 89, 117, and Sooknanan and Malek(1995) Biotechnology 13: 563-564. Improved methods for cloning in vitroamplified nucleic acids are described in Wallace et al., U.S. Pat. No.5,426,039. Improved methods for amplifying large nucleic acids by PCRare summarized in Cheng et al. (1994) Nature 369: 684-685 and thereferences cited therein, in which PCR amplicons of up to 40 kb aregenerated. One of ordinary skill in the art will readily appreciate thatessentially any RNA can be converted into a double stranded DNA suitablefor restriction digestion, PCR expansion and sequencing using reversetranscriptase and a polymerase. See, e.g., Ausubel, Sambrook and Berger,all supra.

Vectors, Promoters, and Expression Systems

The present invention also includes recombinant constructs comprisingone or more of the HHDH polynucleotide sequences as broadly describedabove. The term “construct” or “nucleic acid construct” refers herein toa nucleic acid, either single- or double-stranded, which is isolatedfrom a naturally occurring gene or which has been modified to containsegments of nucleic acids in a manner that would not otherwise exist innature. The term “nucleic acid construct” is synonymous with the term“expression cassette” when the nucleic acid construct contains thecontrol sequences required for expression of an HHDH coding sequence ofthe present invention.

The present invention also provides an expression vector comprising anHHDH polynucleotide of the present invention operably linked to apromoter. Example 1 provides a description of how to make expressionconstructs for expression of halohydrin dehalogenase. The term “controlsequences” refers herein to all the components that are necessary oradvantageous for the expression of a polypeptide of the presentinvention. Each control sequence may be native or foreign to thenucleotide sequence encoding the polypeptide. Such control sequencesinclude, but are not limited to, a leader, polyadenylation sequence,propeptide sequence, promoter, signal peptide sequence, andtranscription terminator. At a minimum, the control sequences include apromoter, and transcriptional and translational stop signals. Thecontrol sequences may be provided with linkers for the purpose ofintroducing specific restriction sites facilitating ligation of thecontrol sequences with the coding region of the nucleotide sequenceencoding a polypeptide.

The term “operably linked” refers herein to a configuration in which acontrol sequence is appropriately placed at a position relative to thecoding sequence of the DNA sequence such that the control sequencedirects the expression of a polypeptide.

When used herein, the term “coding sequence” is intended to cover anucleotide sequence, which directly specifies the amino acid sequence ofits protein product. The boundaries of the coding sequence are generallydetermined by an open reading frame, which usually begins with the ATGstart codon. The coding sequence typically includes a DNA, cDNA, and/orrecombinant nucleotide sequence.

As used herein, the term “expression” includes any step involved in theproduction of the polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

The term “expression vector” refers herein to a DNA molecule, linear orcircular, that comprises a segment encoding a polypeptide of theinvention, and which is operably linked to additional segments thatprovide for its transcription.

As used herein, the term “host cell” refers to any cell type which issusceptible to transformation with a nucleic acid construct.

Nucleic acid constructs of the present invention comprise a vector, suchas, a plasmid, a cosmid, a phage, a virus, a bacterial artificialchromosome (BAC), a yeast artificial chromosome (YAC), or the like, intowhich a nucleic acid sequence of the invention has been inserted, in aforward or reverse orientation. In a preferred aspect of thisembodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available.

Polynucleotides of the present invention can be incorporated into anyone of a variety of expression vectors suitable for expressing apolypeptide. Suitable vectors include chromosomal, nonchromosomal andsynthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids;phage DNA; baculovirus; yeast plasmids; vectors derived fromcombinations of plasmids and phage DNA, viral DNA such as vaccinia,adenovirus, fowl pox virus, pseudorabies, adenovirus, adeno-associatedvirus, retroviruses and many others. Any vector that transduces geneticmaterial into a cell, and, if replication is desired, which isreplicable and viable in the relevant host can be used.

When incorporated into an expression vector, a polynucleotide of theinvention is operatively linked to an appropriate transcription controlsequence (promoter) to direct mRNA synthesis, e.g., T5 promoter.Examples of such transcription control sequences particularly suited foruse in transgenic plants include the cauliflower mosaic virus (CaMV) andfigwort mosaic virus (FMV). Other promoters known to control expressionof genes in prokaryotic or eukaryotic cells or their viruses and whichcan be used in some embodiments of the invention include SV40 promoter,E. coli lac or trp promoter, phage lambda P_(L) promoter, tac promoter,T7 promoter, and the like. An expression vector optionally contains aribosome binding site for translation initiation, and a transcriptionterminator, such as PinII. The vector also optionally includesappropriate sequences for amplifying expression, e.g., an enhancer.

In addition, the expression vectors of the present invention optionallycontain one or more selectable marker genes to provide a phenotypictrait for selection of transformed host cells. Suitable marker genesinclude those coding for resistance to the antibiotic spectinomycin orstreptomycin (e.g., the aada gene), the streptomycin phosphotransferase(SPT) gene coding for streptomycin resistance, the neomycinphosphotransferase (NPTII) gene encoding kanamycin or geneticinresistance, the hygromycin phosphotransferase (HPT) gene coding forhygromycin resistance. Additional selectable marker genes includedihydrofolate reductase or neomycin resistance for eukaryotic cellculture, and tetracycline or ampicillin resistance in E. coli.

An exemplary expression vector for the expression of HHDH polypeptidesof the present invention is depicted in FIG. 1. Vectors of the presentinvention can be employed to transform an appropriate host to permit thehost to express an invention protein or polypeptide. Examples ofappropriate expression hosts include bacterial cells, such as E. coli,B. subtilis, and Streptomyces. In bacterial systems, a number ofexpression vectors may be selected, such as, for example,multifunctional E. coli cloning and expression vectors.

HHDH polynucleotides of the invention can also be fused, for example,in-frame to nucleic acids encoding a secretion/localization sequence, totarget polypeptide expression to a desired cellular compartment,membrane, or organelle of a cell, or to direct polypeptide secretion tothe periplasmic space or into the cell culture media. Such sequences areknown to those of skill, and include secretion leader peptides,organelle targeting sequences (e.g., nuclear localization sequences,endoplasmic reticulum (ER) retention signals, mitochondrial transitsequences, chloroplast transit sequences), membrane localization/anchorsequences (e.g., stop transfer sequences, GPI anchor sequences), and thelike.

Expression Hosts

The present invention also relates to engineered host cells that aretransduced (transformed or transfected) with a vector or construct ofthe invention (e.g., an invention cloning vector or an inventionexpression vector), as well as the production of polypeptides of theinvention by recombinant techniques. The vector may be, for example, aplasmid, a viral particle, a phage, etc. Thus, the present invention isdirected to a host cell comprising any polynucleotide of the presentinvention that is described hereinabove. Typically, the polynucleotideis operably connected to one or more promoters and/or enhancers thatprovide for expression of the polynucleotide of the host cell.

The host cell can be a eukaryotic cell, such as a plant cell.Alternatively, the host cell can be a prokaryotic cell, such as a plantcell. Introduction of the construct into the host cell can be effectedby calcium phosphate transfection, DEAE-Dextran mediated transfection,electroporation, or other common techniques (Davis, L., Dibner, M. andBattey, I. (1986) Basic Methods in Molecular Biology). The engineeredhost cells can be cultured in conventional nutrient media modified asappropriate for activating promoters, selecting transformants, oramplifying the HHDH polynucleotide. Culture conditions, such astemperature, pH and the like, are those previously used with the hostcell selected for expression, and will be apparent to those skilled inthe art and in the references cited herein, including, e.g., Sambrook,Ausubel and Berger, as well as e.g., Freshney (1994) Culture of AnimalCells, a Manual of Basic Technique, third edition, Wiley-Liss, New Yorkand the references cited therein.

HHDH polypeptides of the invention can be produced in non-animal cellssuch as plants, yeast, fungi, bacteria, and the like. In addition toSambrook, Berger and Ausubel, details regarding non-animal cell culturecan be found in Payne et al. (1992) Plant Cell and Tissue Culture inLiquid Systems John Wiley & Sons, Inc. New York, N.Y.; Gamborg andPhillips (eds) (1995) Plant Cell, Tissue and Organ Culture; FundamentalMethods Springer Lab Manual, Springer-Verlag (Berlin Heidelberg NewYork) and Atlas and Parks (eds) The Handbook of Microbiological Media(1993) CRC Press, Boca Raton, Fla. The host cell can be a eukaryoticcell, such as a plant cell. Alternatively, the host cell can be aprokaryotic cell, such as a bacterial cell. Introduction of theconstruct into the host cell can be effected by calcium phosphatetransfection, DEAE-Dextran mediated transfection, electroporation, orother common techniques (Davis, L., Dibner, M., and Battey, I. (1986)Basic Methods in Molecular Biology).

Fusion Polypeptides for Purification

HHDH polypeptides of the present invention may also be expressed as partof a fusion polypeptide to facilitate purification of the encoded HHDHpolypeptide. Polynucleotides encoding such fusion polypeptides comprisea nucleic acid sequence corresponding to an HHDH polynucleotide of thepresent invention that is fused-in frame to a purification facilitatingdomain. As used herein, the term “purification facilitating domain”refers to a domain that mediates purification of the polypeptide towhich it is fused. Suitable purification domains include metal chelatingpeptides, histidine-tryptophan modules that allow purification onimmobilized metals, a sequence which binds glutathione (e.g., GST), ahemagglutinin (HA) tag (corresponding to an epitope derived from theinfluenza hemagglutinin protein; Wilson et al. (1984) Cell, 37:767),maltose binding protein sequences, the FLAG epitope utilized in theFLAGS extension/affinity purification system (Immunex Corp, Seattle,Wash.), and the like. The inclusion of a protease-cleavable polypeptidelinker sequence between the purification domain and the HHDH polypeptideis useful to facilitate purification. One expression vector contemplatedfor use in the compositions and methods described herein provides forexpression of a fusion protein comprising a polypeptide of the inventionfused to a polyhistidine region separated by an enterokinase cleavagesite. The histidine residues facilitate purification on IMIAC(immobilized metal ion affinity chromatography, as described in Porathet al. (1992) Protein Expression and Purification 3:263-281) while theenterokinase cleavage site provides a means for separating the HHDHpolypeptide from the fusion protein. pGEX vectors (Promega; Madison,Wis.) may also be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption to ligand-agarose beads (e.g., glutathione-agarose in thecase of GST-fusions) followed by elution in the presence of free ligand.

Production and Recovery of HHDH Polypeptides

The present invention is directed to a method of making a polypeptidehaving HHDH enzymatic activity, the method comprising providing a hostcell transformed with any one of the described HHDH polynucleotides ofthe present invention; culturing the transformed host cell in a culturemedium under conditions that cause said polynucleotide to express theencoded HHDH polypeptide; and recovering or isolating the expressed HHDHpolypeptide from the culture medium or from the transformed and culturedhost cells.

In another embodiment, the present invention is directed to a method ofmaking a polypeptide having HHDH enzymatic activity, the methodcomprising transforming a host cell with any one of the above describedpolynucleotides of the present invention; culturing the transformed hostcell in a culture medium under conditions that cause said polynucleotideto express the encoded HHDH polypeptide; and recovering or isolating theexpressed HHDH polypeptide from the culture medium or from thetransformed and cultured host cells

The present invention further provides a method of making an HHDHpolypeptide, said method comprising cultivating a host cell transformedwith an HHDH polynucleotide under conditions suitable for the productionof the HHDH polypeptide; and recovering the HHDH polypeptide.

Typically, recovery or isolation is from the host cell culture medium,the host cell or both, using protein recovery techniques that are wellknown in the art, including those described below.

Following transduction of a suitable host strain and growth (cultivatingor culturing) of the host strain to an appropriate cell density, theselected promoter is induced by appropriate means (e.g., temperatureshift or chemical induction) and cells are cultured for an additionalperiod. As used herein, the terms “culturing” and “cultivating” are usedinterchangeably. Cells are typically harvested by centrifugation,disrupted by physical or chemical means, and the resulting crude extractretained for further purification. Microbial cells employed inexpression of proteins can be disrupted by any convenient method,including freeze-thaw cycling, sonication, mechanical disruption, or useof cell lysing agents, or other methods, which are well known to thoseskilled in the art.

As noted, many references are available for the culture and productionof many cells, including cells of bacterial, plant, animal (especiallymammalian) and archebacterial origin. See e.g., Sambrook, Ausubel, andBerger (all supra), as well as Freshney (1994) Culture of Animal Cells,a Manual of Basic Technique, third edition, Wiley-Liss, New York and thereferences cited therein; Doyle and Griffiths (1997) Mammalian CellCulture: Essential Techniques John Wiley and Sons, NY; Humason (1979)Animal Tissue Techniques, fourth edition W.H. Freeman and Company; andRicciardelli, et al., (1989) In vitro Cell Dev. Biol. 25:1016-1024. Forplant cell culture and regeneration, Payne et al. (1992) Plant Cell andTissue Culture in Liquid Systems John Wiley & Sons, Inc. New York, N.Y.;Gamborg and Phillips (eds) (1995) Plant Cell, Tissue and Organ Culture;Fundamental Methods Springer Lab Manual, Springer-Verlag (BerlinHeidelberg New York); Jones, ed. (1984) Plant Gene Transfer andExpression Protocols, Humana Press, Totowa, N.J. and Plant MolecularBiology (1993) R. R. D. Croy, Ed. Bios Scientific Publishers, Oxford,U.K. ISBN 0 12 198370 6. Cell culture media in general are set forth inAtlas and Parks (eds.) The Handbook of Microbiological Media (1993) CRCPress, Boca Raton, Fla. Additional information for cell culture is foundin available commercial literature such as the Life Science ResearchCell Culture Catalogue (1998) from Sigma-Aldrich, Inc (St Louis, Mo.)(“Sigma-LSRCCC”) and, e.g., The Plant Culture Catalogue and supplement(1997) also from Sigma-Aldrich, Inc (St Louis, Mo.) (“Sigma-PCCS”).

HHDH polypeptides of the present invention can be recovered/isolated andoptionally purified from recombinant cell cultures by any of a number ofmethods well known in the art, including ammonium sulfate or solvent(e.g., ethanol, acetone, and the like) precipitation, acid extraction,anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography (e.g., using any of the tagging systems noted herein),hydroxylapatite chromatography, and lectin chromatography. Proteinrefolding steps can be used, as desired, in completing the configurationof the mature protein. Finally, high performance liquid chromatography(HPLC) can be employed in the final purification steps. In addition tothe references noted supra, a variety of purification methods are wellknown in the art, including, e.g., those set forth in Sandana (1997)Bioseparation of Proteins, Academic Press, Inc.; Bollag et al. (1996)Protein Methods, 2^(nd) Edition, Wiley-Liss, NY; Walker (1996) TheProtein Protocols Handbook Humana Press, NJ; Harris and Angal (1990)Protein Purification Applications: A Practical Approach, IRL Press atOxford, Oxford, England; Harris and Angal Protein Purification Methods:A Practical Approach, IRL Press at Oxford, Oxford, England; Scopes(1993) Protein Purification: Principles and Practice 3^(rd) Edition,Springer Verlag, NY; Janson and Ryden (1998) Protein Purification:Principles, High Resolution Methods and Applications, Second EditionWiley-VCH, NY; and Walker (1998) Protein Protocols on CD-ROM, HumanaPress, NJ.

In some cases, it may be desirable to produce the HHDH polypeptides ofthe invention on a large scale suitable for industrial and/or commercialapplications. In such cases, bulk fermentation procedures are employed.An exemplary bulk fermentation procedure for producing HHDH is providedin Example 2. Briefly, an HHDH polynucleotide is cloned into anexpression vector, such as, for example, the vector depicted in FIG. 1(PCK110700). After inserting the polynucleotide of interest into avector, the vector is tranformed into a bacterial host, such as, forexample, E. coli BL21 (Strategene, La Jolla, Calif.) after passagethrough E. coli TOP10 (Invitrogen, Carlsbad, Calif.) using standardmethods.

The transformed cells are cultivated in a nutrient medium suitable forproduction of the polypeptide using methods that are known in the art.For example, the cell may be cultivated by shake flask cultivation,small-scale or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors performed in a suitable medium and under conditions allowingthe polypeptide to be expressed and/or isolated. The cultivation takesplace in a suitable nutrient medium comprising carbon and nitrogensources and inorganic salts, using procedures known in the art. Suitablemedia are available from commercial suppliers or may be preparedaccording to published compositions (e.g., in catalogues of the AmericanType Culture Collection). The secreted polypeptide can be recovereddirectly from the nutrient (culture) medium.

The resulting polypeptide may be isolated by methods known in the art.For example, the polypeptide may be isolated from the nutrient medium byconventional procedures including, but not limited to, centrifugation,filtration, extraction, spray-drying, evaporation, or precipitation. Theisolated polypeptide may then be further purified by a variety ofprocedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), or extraction (see, e.g., Bollag et al.(1996) Protein Methods, 2^(nd) Edition, Wiley-Liss, NY; Walker (1996)The Protein Protocols Handbook, Humana Press, NJ; and Bollag et al.(1996) Protein Methods, 2^(nd) Edition, Wiley-Liss, NY. A procedure forrecovering the HHDH polypeptide from a cell lysate is illustrated inExample 3.

Cell-free transcription/translation systems can also be employed toproduce HHDH polypeptides using the polynucleotides of the presentinvention. Several such systems are commercially available. A generalguide to in vitro transcription and translation protocols is found inTymms (1995) In vitro Transcription and Translation Protocols: Methodsin Molecular Biology, Volume 37, Garland Publishing, NY.

Methods of Using HHDH Polypeptides

As described supra, HHDH polypeptides of the present invention can beused to catalyze the conversion of 4-halo-3-hydroxybutyric acidderivatives to 4-nucleophile substituted-3-hydroxybutyric acidderivatives. Thus, the present invention provides a method for producingethyl (R) 4-cyano-3-hydroxybutyrate, said method comprising: (a)providing a 4-halo-3-hydroxybutyric acid ester, wherein the halosubstituent is selected from the group consisting of chlorine, bromine,and iodine; and (b) contacting the 4-halo-3-hydroxybutyric acid esterwith a halohydrin dehalogenase and cyanide under conditions sufficientto form a reaction mixture for converting the 4-halo-3-hydroxybutyricacid ester to a 4-cyano-3-hydroxybutyric acid ester. A suitable4-halo-3-hydroxybutyric acid ester/4-cyano-3-hydroxybutyric acid estercombination is ethyl (S)-4-chloro-3-hydroxybutyrate and ethyl (R)4-cyano-3-hydroxybutyrate. The method is described in Internationalpublication WO 2004/015132, “Enzymatic Processes for the Production of4-Substituted 3-Hydroxybutyric Acid Derivatives,” which is incorporatedherein by reference. A description of conditions suitable for convertingethyl (S)-4-chloro-3-hydroxybutyrate to ethyl(R)_-4-cyano-3-hydroxybutyrate and recovering the latter can also befound in International publication WO 2004/015132, “Enzymatic Processesfor the Production of 4-Substituted 3-Hydroxybutyric Acid Derivatives,”which is incorporated herein by reference.

HHDH polypeptides of the present invention are also useful forcatalyzing the conversion of a vicinal halo (i.e., chlorine, bromine, oriodine), hydroxy-substituted carboxylic acid ester to a vicinal cyano,hydroxyl substituted carboxylic acid ester. Accordingly, the presentinvention also provides a method for producing a vicinal cyano, hydroxysubstituted carboxylic acid ester from a vicinal halo, hydroxylsubstituted carboxylic acid ester, the method comprising: (a) providinga vicinal halo, hydroxyl substituted carboxylic acid ester, wherein thehalo substituent is selected from the group consisting of chlorine,bromine, and iodine; (b) contacting the vicinal halo-hydroxylsubstituted carboxylic acid ester with an HHDH polypeptide of thepresent invention and cyanide under conditions suitable to form areaction mixture for converting the vicinal halo, hydroxy substitutedcarboxylic acid ester to a vicinal cyano, hydroxy substituted carboxylicacid ester.

This method is described in International Application WO 2005/01859,“Enzymatic Processes for the Production of 4-Substituted3-Hydroxybutyric Acid Derivatives and Vicinal Cyano, Hydroxy SubstitutedCarboxylic Acid Esters,” which is incorporated herein by reference. Thisreference provides a description of conditions suitable for converting avicinal halo, hydroxyl substituted carboxylic acid ester to a vicinalcyano, hydroxyl substituted carboxylic acid ester.

The novel halohydrin dehalogenases of the present invention are alsouseful in the process for enzymatically resolving a mixture ofenantiomeric epoxides by reacting the mixture with an anionicnucleophile in the presence of the halohydrin dehalogenase, wherein theenzyme preferentially reacts one of the epoxide enantiomers with thenucleophile to form a mixture of the resulting enantiomerically enrichedvicinal nucleophile-substituted alcohol and the unreacted epoxideenriched in the other enantiomer, in the manner disclosed in publicationWO 01/90397, which is incorporated herein by reference in its entirety.

The foregoing and other aspects of the invention may be betterunderstood in connection with the following non-limiting examples.

EXAMPLES Example 1 Construction of Expression Constructs for Expressionof Halohydrin Dehalogenase

The gene for Agrobacterium sp. halohydrin dehalogenase was codonoptimized (SEQ ID NO: 1) for expression in E. coli based on the aminoacid sequence of the halohydrin dehalogenase from Agrobacterium sp. (SEQID NO: 2). The gene was synthesized using 60-mer oligomers, and clonedinto expression vector PCK110700 (depicted in FIG. 1) under the controlof a T5 promoter. The vector was transformed into E. coli TOP10(Invitrogen, Carlsbad, Calif.) from which plasmid DNA was prepared usingstandard methods. The plasmid DNA was then transformed into E. coli BL21(Stratagene, La Jolla, Calif.), the expression host, using standardmethods. A clone was found in the expression library that expressedactive HHDH. The gene from this clone was sequenced (see SEQ ID NO: 1(HHDH.1)) and found to encode Agrobacterium sp. HHDH (SEQ ID NO: 2).

Polynucleotides encoding halohydrin dehalogenases of the presentinvention were similarly cloned into vector PCK 110700, depicted in FIG.1, then transformed and expressed from E. coli BL21 after passagethrough E. coli TOP10 using standard methods.

Example 2 Production of HHDH

In an aerated agitated fermentor, 10.0L of growth medium containing0.528 g/L ammonium sulphate; 7.5 g/L of di-potassium hydrogen phosphatetrihydrate; 3.7 g/L of potassium dihydrogen phosphate; 2 g/L ofTastone-154 yeast extract; 0.05 g/L ferrous sulphate; and 3 ml/L of atrace element solution containing 2 g/L of calcium chloride dihydrate,2.2 g/L of zinc sulfate septahydrate, 0.5 g/L manganese sulfatemonohydrate, 1 g/L cuprous sulfate heptahydrate: 0.1 g/L sodium boratedecahydrate and 0.5 g/L EDTA, was brought to a temperature of 30° C. Thefermentor was inoculated with a late exponential culture of Escherichiacoli BL21 (Stratagene, La Jolla, Calif.) equipped with plasmidcontaining HHDH polynucleotides as described in Example 1, then grown ina shake flask containing LB, 1% glucose (Sigma Chemical Co., St. Louis,Mo.), and 30 μg/ml chloroamphenicol (Sigma Chemical Co., St. Louis, Mo.)to a starting optical density at 600 nm (OD₆₀₀) of 0.5 to 2.0. Thefermentor was agitated at 500-1500 rpm and air was supplied to thefermentation vessel at 1.0-15.0 L/min to maintain a dissolved oxygenlevel of 30% saturation or greater. The pH of the culture was controlledat 7.0 by addition of 20% v/v ammonium hydroxide. After the culturereached an OD₆₀₀ of 40, the temperature was maintained at 30° C. and theexpression of halohydrin dehalogenase was induced by the addition ofisopropyl-β-D-thiogalactoside (IPTG) (Sigma Chemical Corp., St. Louis,Mo.) to obtain a final concentration of 1 mM. The culture was grown foranother 15 hours. After the induction, the cells were harvested bycentrifugation and washed with 10 mM potassium phosphate buffer, pH 7.0.The cell paste was used directly in the downstream recovery process orwas stored at −80° C. until use.

Example 3 Enzyme Preparation

The cell paste from Example 2 was washed by suspending 1 volume wetweight of cell paste in 3 volumes of 100 mM Tris/sulfate (pH 7.2)followed by centrifugation at 5000 g for 40 minutes in a Sorval 12BP.The washed cell paste was suspended in 2 volumes of 100 mM Tris/sulfate(pH 7.2). The intracellular HHDH was released from the cells by passingthe suspension through a homogenizer in two passes using a pressure of14,000 psig for the first pass and 8,000 psig for the second pass. Thecell lysate was allowed to cool to 4° C. between passes through thehomogenizer. The lysate is warmed to room temperature and then either2.5M MnSO₄ (50-350 mM final concentration), or a 10% w/v solution ofpolyethyleneimine (PEI), pH 7.2, (0.6-1.0% w/v final concentration) wasadded to the lysate and stirred for 30 minutes. The homogenate wascentrifuged at between 5,000 and 10,000 g in a standard laboratorycentrifuge for 30 to 60 minutes. The supernatant was desalted,concentrated by ultrafiltration, dispensed in shallow containers, frozenat −20° C. and lyophilized to a powder that was stored at −80° C.

To assess the quality of the preparation after fermentation, cell lysatecontaining the expressed halohydrin dehalogenase enzyme was assayedaccording to the following protocol. Approximately 50 μl of clarifiedcell lysate in 10 mM Tris-SO₄, 100 mM NaCN, pH 8.0 was mixed with 10 mMethyl-(S)-4-chloro-3-hydroxybutyrate (ECHB) (Sigma Aldrich, St. Louis,Mo.). The total reaction volume was 0.2 ml. The reaction was incubatedat room temperature for 30 min to 1 hour. The reaction was extractedwith 7 volumes of ethyl acetate and the organic layer removed to a 1.8ml gas chromatography (GC) vial. The organic layer was analyzed by GCfor presence of the ethyl-(R)-4-cyano-3-hydroxybutyrate product. Theamount of product produced was determined by comparison to a standardcurve prepared and analyzed under the same conditions.

Example 4 High Throughput Screen for Presence of HHDH Activity

A. No Cyanohydrin in Agarose

The following screen was used to ascertain the presence of HHDHactivity. On day 1, freshly transformed colonies on a Q-tray (GenetixUSA, Inc. Beaverton, Oreg.) containing 200 ml LB agar+1% glucose, 30μg/ml chloramphenicol were picked using a Q-bot® robot colony picker(Genetix USA, Inc., Beaverton, Oreg.) into shallow 384 well Nunc platescontaining media (70 μL/well 2xYT+1% glucose, 30 μg/ml chloramphenicol)(Nalge Nunc International, Rochester, N.Y.) for overnight growth at 30°C., 250 revolutions per minute (rpm), 85% relative humidity (RH). Anegative control (E. coli BL21 with empty vector) and a positive control(E. coli BL21 with vector containing HHDH Mz1/2G5, SEQ ID NO: 31) wereincluded. These master well plate cultures were covered with AirPore™microporous tape (Qiagen, Inc., Valencia, Calif.).

On day 2, the master plate cultures were gridded onto nylon membranes(Pall Biodyne B Nylon Membrane pre-cut for Omnitray, 115×76 mm, NalgeNunc #250385) then placed onto a Q-tray (Genetix USA, Inc. Beaverton,Oreg.) containing 200 ml LB agar+1% glucose, 30 μg/ml chloramphenicol.The Q-trays were incubated at 30° C. for 8-12 hours until growth wasdetected. Each nylon membrane was transferred to a Q-tray containinginducing media: 200 ml LB agar+1 mM IPTG, 30 μg/ml chloramphenicol. TheQ-trays were then incubated at 23° C. or room temperature overnight.

On day 3, the assay plate was prepared as follows: a solution of 150 mlof 10 mM Tris-SO₄, pH 7.0, and 1.0% low melt agarose was prepared andcooled to about 45° C. 5M NaCl was added to give a final concentrationof 500 mM NaCl. Bromcresol purple (BCP) and ethyl(S)-4-chloro-3-hydroxybutyrate (ECHB) were added to final concentrationsof 0.004% and 0.3%, respectively. The solution was poured into a 150 mlQ-tray and allowed to solidify.

The nylon membrane with the colonies was removed from the Q traycontaining inducing media and inverted onto the assay plate. Themembrane was imaged through the inverted Q-tray using the Alpha ImagingChemStation (Alpha Innotech Corporation, San Leandro, Calif.), aperturesetting of 4 with a 420 nm (+/−10 nm filter). An image was acquiredduring the first hour of the reaction. The intensity data for eachimaged spot was then normalized to the value of the negative controlspots. A normalized value greater than one indicated the presence ofHHDH activity. Active clones from this screen were further characterizedusing the method described in Example 5A. Clones from this screen mayalso be further characterized using the medium throughput assaydescribed in Example 5B.

B. Cyanohydrin in Agarose

This high throughput screen is used when it is desired to screen forHHDH polypeptides that exhibit HHDH activity in the presence ofcyanohydrin product, e.g., ethyl (R)-4-cyano-3-hydroxybutyrate. Theprotocols for days one and two are the same as recited in part A. On day3, the assay plate was prepared as follows: a 150 ml low melt agarosesolution was made up as follows: 10 mM Tris, pH 7.0, 2.0% low meltagarose (melted in microwave), 0.004% bromcresol purple (1.2 ml/150 ml).The solution was cooled to 37° C. overnight. On day three, ECHB (0.45 mlECHB/150 ml solution) and ethyl (R)-4-cyano-3-hydroxybutyrate (8.26 mlethyl (R)-4-cyano-3-hydroxybutyrate/150 ml solution) were added to givea 0.3% ECHB and 400 mM ethyl (R)-4-cyano-3-hydroxybutyrate solution. Thesolution was mixed and poured into a 150 ml Q-tray, then allowed tosolidify as described in part A.

The nylon membrane with the colonies was removed from the Q traycontaining the inducing media and inverted onto the assay plate. Themembrane was imaged as described in part A above.

Active clones from this screen were further characterized using the gaschromatography method described in Example 5B (Medium through-putassay).

Example 5 Characterization of Halohydrin Dehalogenase Activity

A. Gas Chromatography Method for Detection of ProductEthyl-(R)-4-cyano-3-hydroxybutyrate

To a solution of ethyl (S)-4-chloro-3-hydroxybutyrate (10 mM-100 mM) in500 mM HCN (500 mM NaCN adjusted to pH 7.0 with phosphoric acid) wasadded the halohydrin dehalogenase enzyme as a predissolved solution inthe same buffer. Over time, aliquots of the mixture were withdrawn andextracted with three volumes of ethyl acetate. The organic layer wasthen analysed for ethyl (R)-4-cyano-3-hydroxybutyrate by gaschromatography (GC), as described hereinbelow in Example 6. Samples weretaken at various time points, and the peak area of the productcyanohydrin, ethyl (R)-4-cyano-3-hydroxybutyrate, was plotted as afunction of time. Time points are selected at low conversion, forexample, less than 5% conversion, to avoid the effect of productinhibition (e.g., 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, etc.). The peakareas were converted to concentration units using a standard curve thatwas prepared for the ethyl (R)-4-cyano-3-hydroxybutyrate. Activity ofthe halohydrin dehalogenase was determined in units of μmol (cyanohydrinproduced)/min/mg (total halohydrin dehalogenase catalyst). Relativeactivities of some of the clones are shown in Table 2, computed asActivity of Improved HHDH Enzyme/Activity of Agrobacterium sp. HHDH (SEQID NO: 2).

TABLE 2 Relative HHDH Activity of Improved HHDH Enzymes on ECHBSubstrate Fold Improvement in HHDH Activity over SEQ ID NO:Agrobacterium sp. HHDH (SEQ ID NO: 2) (SEQ ID NO: 4) 1.5 (SEQ ID NO: 6)1.6 (SEQ ID NO: 8) 1.8 (SEQ ID NO: 10) 1.7 (SEQ ID NO: 34) 2.4 (SEQ IDNO: 12) 2.5 (SEQ ID NO: 14) 1.4 (SEQ ID NO: 16) 2.0 (SEQ ID NO: 18) 2.7(SEQ ID NO: 20) 3.8 (SEQ ID NO: 22) 2.5 (SEQ ID NO: 24) 3.2 (SEQ ID NO:26) 1.7 (SEQ ID NO: 28) 2.2 (SEQ ID NO: 30) 2.8B. Medium Throughput-Gas Chromatography Assay in Presence of CyanohydrinProduct

Hits were picked from desired wells (10 μL of culture) in the prescreenmaster well plates and transferred into the wells of 96 well NUNC plates(each well containing 200 ul LB+1% glucose, 30 μg/ml chloramphenicol(cam)) for overnight growth at 30° C., 250 rpm, 85% relative humidity.The positive controls were picked from the prescreen master well plates.

The next day, 10 μl aliquots of the overnight growth was subculturedinto 96 deep well plates each well containing 300 μl 2xYT, 100 mMNaH₂PO₄/Na₂HPO₄ pH 7, 1 mM MgSO₄, 30 μg/ml cam. These plates wereincubated at 30° C., 250 rpm, 85% relative humidity, 24 hrs, until thecell density reached an OD 600 nm=0.6. The plates were then induced with1 mM isopropyl-β-D-thiogalactoside (IPTG) (e.g., 10 μl/well of a 34 mMIPTG stock solution or 30 uL/well of 10 mM IPTG stock) and incubated at30° C. overnight, 250 rpm, 85% relative humidity.

The next day, the plates were centrifuged (4000 rpm, 10 min., 4° C.) topellet the cells and the spent media was discarded. The plates can befrozen at −80° C. for one hour to aid in cell breakage.

The pelleted cells were lysed by adding 200 μL B-PER® lysing solution(Pierce, cat# 78243) containing 2.04 M ethyl-4-cyano-3-hydroxybutyrate(“HN”) (320 g/L)(fw=157, d 1.19, 26.8 ml/100 ml lysis mixture) and 1ul/10 ml DNase (˜200 U/ul). The mixture of cells and lysing solution wasvortexed to resuspend the cells and then incubated at 50° C. withshaking for two hours.

A reaction solution was made up in a fume hood, preferably using aplastic (polypropylene) disposal container. The volume of reactionsolution was determined by number of plates screened. To prepare thereaction solution having a 1M final concentration of NaCN, NaCN(fw=49.01, 4.9 g/100 mL) was added to the desired volume of 100 mMsodium phosphate pH 7 to give a 1.47M concentration of NaCN. To each 68mL of the NaCN solution was added 24 mL of 5M stock NaCl and 8 ml ofconcentrated HCl (˜10 M) to produce the desired volume of reactionmixture that was 1.2 M NaCl, 800 mM HCl, and 1M NaCN. The final pH ofthe reaction mixture was 7.0-7.2. To this solution was added ECHB(fw=166.6, d=1.19) at 280 μL/100 mL reaction mix to obtain a 20 mM finalconcentration. The final concentrations in the reaction mix are ˜1M HCN,2M NaCl, 50 mM sodium phosphate pH 7.0 to 7.2, 20 mM ECHB.

200 μL of the reaction mixture was added to the lysed cells in eachwell. The plates were sealed using the Velocity11 PlateLoc™ heat sealer.The sealed plates were then shaken at room temperature for 120 minutes.After shaking, the plates were unsealed and 1 mL of 1 mM thymol(dissolved in ethyl acetate) was added to each well. The plates wereresealed using the Velocity11 PlateLoc™ heat sealer, shaken vigorously,then allowed to sit for ˜1 minute to let the layers separate.

150 μL aliquots of the upper layer were transferred to Costar roundbottom shallow well polypropylene (PP) reaction plates (Cat# 3365) usinga Hydra™ positive displacement liquid handler (Asp mode, AV 150, AH2650, EH 37800, WH 3730, WV full, Wash 3). Samples were transferred fromthe deep well plate into the shallow well plates.

These plates were sealed using the Velocity11 PlateLoc™ heat sealer andstored at −20° C. until analysis by Gas Chromatography as described inExample 6B.

C. Medium Throughput-Gas Chromatography Assay for Inhibition in thePresence of Ethyl-4-Chloroacetoacetate (ECAA)

Hits were picked from desired wells (10 μL of culture) in the prescreenmaster well plates and transferred into the wells of 96 well NUNC plates(each well containing 200 ul LB+1% glucose, 30 μg/ml chloramphenicol(cam)) for overnight growth at 30° C., 250 rpm, 85% relative humidity.The positive controls were picked from the prescreen master well plates.

The next day, 10 μl aliquots of the overnight growth was subculturedinto 96 deep well plates, each well containing 300 μl 2xYT, 100 mMNaH₂PO₄/Na₂HPO₄ pH 7, 1 mM MgSO₄, 30 μg/ml cam. These plates wereincubated at 30° C., 250 rpm, 85% relative humidity, 2-4 hrs, until thecell density reached an OD 600 nm=0.6. The plates were then induced with1 mM IPTG (e.g., 10 μl/well of a 34 mM IPTG stock solution or 30 μL/wellof 10 mM IPTG stock) and incubated at 30° C. overnight, 250 rpm, 85%relative humidity.

The next day, the plates were centrifuged (4000 rpm, 10 min., 4° C.) topellet the cells and the spent media was discarded. The plates can befrozen at −80° C. for one hour to aid in cell breakage.

The pelleted cells were lysed by adding 200 μL B-PER® lysing solution(Pierce, cat# 78243) with 1 ul/10 ml DNase (˜200 U/ul). The mixture ofcells and lysing solution was vortexed to resuspend the cells and thenincubated at 50° C. with shaking for two hours.

A reaction solution was made up in a fume hood, preferably using aplastic (PP) disposal container (volume determined by number of platesscreened). To prepare the reaction solution having a 1M finalconcentration of NaCN, NaCN (fw=49.01, 4.9 g/100 mL) was added to thedesired volume of 100 mM sodium phosphate pH 7 to give 1.47Mconcentration of NaCN. To each 68 mL of the NaCN solution was added 24mL of 5M stock NaCl and 8 ml of concentrated HCl (˜10 M) to produce thedesired volume of reaction mixture that was 1.2 M NaCl, 800 mM HCl, and1M NaCN. The final pH of the reaction mixture is 7.0-7.2. To thissolution was added ECHB (fw=166.6, d=1.19) to 100 mM final concentration(1400 μL/100 mL reaction mix) and ECAA (fw=164.6, d=1.21) to 5 mM finalconcentration (100 μL/100 mL reaction mix).

200 μL of the reaction mixture was added to the lysed cells in eachwell. The plates were sealed using the Velocity11 PlateLoc™ heat sealer.The sealed plates were then shaken at room temperature for 60 minutes.After shaking, the plates were unsealed and 1 mL of 1 mM thymol(dissolved in ethyl acetate) was added to each well. The plates wereresealed using the Velocity11 PlateLoc™ heat sealer, shaken vigorously,then allowed to sit for ˜1 minute to let the layers separate.

150 μl aliquots of the upper layer were transferred to Costar roundbottom shallow well polypropylene (PP) reaction plates (Cat# 3365) usinga Hydra™ positive displacement liquid handler (Asp mode, AV 150, AH2650, EH 37800, WH 3730, WV full, Wash 3). Samples were transferred fromthe deep well plate into the shallow well plates. These plates weresealed using the Velocity11 PlateLoc™ heat sealer and stored at −20° C.until analysis by Gas Chromatography as described in Example 6B.

D. Third Tier Screen of HHDH Activity of HHDH Polypeptides PreparedUnder Conditions of Catalyst Manufacture and Use:

To a 100 mL jacketed round bottom flask equipped a septum, magnetic stirbar and a pH electrode connected to a pH stat, was charged NaCN (0.75 g,15 mmol) followed by deionized water (25 mL). The vessel was sealed andthe pH was adjusted to 7.6 using conc. H₂SO₄ (˜0.5 mL). The HHDH powder(produced as described in Examples 2 (paragraph 193) and 3 (paragraph194) above) was charged as an aqueous solution (75 mg in 5 mL deionizedwater) and the reaction mixture was heated to 40° C. The HHDHpolypeptide used in this assay may be in lysed or purified form andprepared according to methods known in the art. When comparing theperformance of one or more HHDH polypeptides, they should be preparedaccording to the same method. After heating the reaction mixture to 40°C., ethyl S 4-chloro-3-hydroxybutyrate (ECHB, 5 g, 30 mmol) was addedvia syringe. The pH stat maintained the pH at 7.3+/−0.1 with theaddition of 25% NaCN with 0.25% NaOH. The progress of the reaction wasmonitored by removal of 0.1 mL of reaction mix, to which was added ethylacetate (1 mL). The mixture was vortexed and the phases allowed toseparate. The upper, organic layer was analysed by GC (Example 6A). Thereaction was deemed complete when the peak area ratio of ECHB to HN was1/200 as measured by GC in accordance with the method of Example 6A. Thetime period from when all reagents are added to the reaction mixture tothe time when the reaction is deemed complete (peak area ratio of ECHBto HN of 1/200) is determined. This protocol can be used to determinerelative activity in terms of fold improvement relative to a referenceHHDH polypeptide. When determining relative activity, the times to reacha peak area ratio of 1/200, ECHB to HN, are compared, i.e.,t_(peak area 1:200, ECHB:HN of HHDH polypeptide)/t_(peak area 1:200, ECHB:HN of REFERENCE HHDH polypeptide.)(HHDH activity may also be determined from this data in units of μmol(cyanohydrin produced)/min/mg (total halohydrin dehalogenase catalyst)).Many of the HHDH polypeptides described herein exhibit activity of1.5-fold greater than the HHDH polypeptide of SEQ ID NO: 730.

Example 6 A. Detection of Ethyl (R)-4-cyano-3-hydroxybutyrate by GasChromotography

The ethyl (R)-4-cyano-3-hydroxybutyrate produced in Example 5A wasanalyzed using gas chromatography with flame ionization (FID) detectionusing an Agilent® HP-5™ column, 30 m long, 0.32 mm inner diameter, film0.25 μm, using the following program: 1 minute at 100° C., 5° C./minutefor 10 minutes; 25° C./minute for 2 minutes; then 2 minutes at 200° C.Inlet and outlet temperatures were both 300° C., and the flow rate was 2ml/minute. Under these conditions, ethyl (R)-4-cyano-3-hydroxybutyrateelutes at 6.25 minutes and ethyl (S)-4-chloro-3-hydroxybutyrate elutesat 4.5 minutes. Chemical purity of the species was measured using theintegrated peak areas from the gas chromatography results.

Enantioselectivity of the halohydrin dehalogenase (HHDH) with respect toethyl (R)-4-cyano-3-hydroxybutyrate was measured by gas chromatographyand FID detection using a Restek gammaDex SA™ column (30 m long, 0.32 μminner diameter) using the following program: 25 minutes at 165° C. andflow rate at 2 ml/min. Inlet and outlet temperatures were both at 230°C. Under these conditions ethyl (R)-4-cyano-3-hydroxybutyrate elutes at19.6 minutes and ethyl (S)-4-cyano-3-hydroxybutyrate elutes at 19.2minutes.

B. Detection of Remaining Ethyl (S)-4-chloro-3-hydroxybutyrate by GasChromatography

Halohydrin dehalogenases of the present invention that exhibitedactivity in the presence of cyanohydrin product in the prescreen methodof Example 4B, were further characterized in the assay described inExample 5B. The remaining ethyl (S)-4-chloro-3-hydroxybutyrate in thereaction mixture from Example 5B was analyzed using gas chromatographywith an Agilent® 19091J-413 HP-5™ 5% phenyl methyl siloxane column, 30.0m long×320 μm inner diameter×0.25 μm nominal, and a flow rate of 2.6ml/min. The following program was used: 1 minute at 100° C., 50°C./minute for 2 minutes, 2 minutes hold, with a 10 minute cycle time.The detector conditions were as follows: 300° C., 40 ml/min H₂, 450ml/min air. Under these conditions, ethyl (S)-4-chloro-3-hydroxybutyrateelutes at 3.12 minutes, ethyl (R)-4-cyano-3-hydroxybutyrate elutes at3.06 minutes, and thymol elutes at 3.21 minutes. Activity may becharacterized by the quantity of ethyl (S)-4-chloro-3-hydroxybutyrateremaining normalized to the extraction efficiency, i.e., Area ECHB/AreaThymol. Thymol is used as an internal standard for extraction efficiencyof the reaction components from water to ethyl acetate.

Example 7 Manufacture of Ethyl (R)-4-cyano-3-hydroxybutyrate from Ethyl(S)-4-chloro-3-hydroxybutyrate

To a 3-necked jacketed 3L flask equipped with a mechanical stirrer andconnected to an automatic titrater by a pH electrode and a feeding tubefor addition of base, was charged H₂O (1200 mL), NaCN (37.25 g) andNaH₂PO₄ (125 g) to bring the solution to pH 7. The water circulator wasset to 40° C. After 10 minutes, halohydrin dehalogenase of SEQ ID NO: 32as cell lysate (250 mL) was added. The reaction mixture was allowed tostir for 5 minutes. Using an addition funnel, ethyl(S)-4-chloro-3-hydroxybutyrate (45 g) was slowly added over 1 hour. ThepH was maintained at 7 by the automatic titrater by the addition of 10 MNaOH (27 mL) over 17 hours. Subsequently, gas chromatography of areaction sample showed complete conversion to product. Celite™ (16 g)was added to the flask, which was then connected to a diaphragm pump,whose exhaust is bubbled into 5M NaOH (200 mL), to remove HCN. Themixture was heated to 60° C. under 100 mm Hg pressure. After 1 hour, asubmerged air bubbler was added to the solution to aid the removal ofthe HCN. After 3 hours, an HCN detector indicated less than 5 ppm HCN inthe off-gas. The mixture was allowed to cool to room temperature, thenfiltered through a Celite™ pad. The filtrate was extracted with butylacetate (3×800 mL) and the combined organic layers filtered through apad of activated charcoal. The solvent was removed under vacuum byrotary evaporation to provide 28.5 g of ethyl(R)-4-cyano-3-hydroxybutyrate. The purity was 98% (w/w) by HPLC and theenantiomeric excess was >99% (by chiral GC, the S enantiomer wasundetectable). As used herein, the term “enantiomeric excess” or “e.e.”refers to the absolute difference between the mole or weight fractionsof major (F₍₊₎) and minor (F⁽⁻⁾) enantiomers (i.e., |F₍₊₎−F⁽⁻⁾|), whereF₍₊₎+F⁽⁻⁾=1. Percent e.e. is 100×|F₍₊₎−F⁽⁻⁾|. Enantiomeric compositioncan be readily characterized by using the gas chromatography methoddescribed in Example 6, above, and using methods that are known in theart.

Examples 8-12 Conversion of Ethyl (R)-4-chloro-3-hydroxybutyrate toEthyl (S)-4-cyano-3-hydroxybutyrate

For each of Examples 8-12, to a 170 mL vessel connected to an automatictitrater by a pH electrode and a feeding tube for addition of base wascharged NaCN (1.5 g, 31 mmol) and water (50 mL). The vessel was sealedand the pH was adjusted to 7 by the addition of conc. H₂SO₄ (0.9 mL).The reaction mixture was heated to 40° C. and treated with a solution ofhalohydrin dehalogenase (0.4 g in 10 mL water). The halohydrindehalogenases used for these Examples had the polypeptide sequencesgiven for the following SEQ ID NOs:

Example 8 SEQ ID No: 32

Example 9 SEQ ID No: 90

Example 10 SEQ ID No: 94

Example 11 SEQ ID No: 96

Example 12 SEQ ID No: 98

Then, ethyl (S)-4-chloro-3-hydroxybutyrate (5.00 g, 30.1 mmol) was addedvia syringe. The automatic titrater maintained the pH at 7 by theaddition of 4M NaCN. The progress of the reactions was monitored byrecording the cumulative volume of the NaCN solution added vs. time.

FIG. 2 shows the percent conversion of ethyl(S)-4-chloro-3-hydroxy-butyrate (calculated from the cumulativeequivalents of NaCN added) vs. time for each of these Examples. Example8 used a halohydrin dehalogenase having the amino acid sequence SEQ IDNO. 32, which is the amino acid sequence of the native halohydrindehalogenase from Agrobacterium radiobacter AD1 (hheC), expressed fromnovel nucleic acid corresponding to SEQ ID NO. 31. Comparison of thepercent conversion vs. time for Examples 9 through 12 to that of Example8 shows that novel halohydrin dehalogenases of the present inventionhave greater activity than the native halohydrin dehalogenase fromAgrobacterium radiobacter AD1 (hheC).

All publications, patents, patent applications, and other documentscited in this application are incorporated by reference in theirentirety for all purposes to the same extent as if each individualpublication, patent, patent application, or other document wereindividually indicated to be incorporated by reference for all purposes.

While preferred embodiments of the invention have been illustrated anddescribed, it will be readily appreciated that various changes can bemade therein without departing from the spirit and scope of theinvention.

1. A recombinant Halohydrin dehalogenase (HHDH) polypeptide capable ofconverting ethyl (S)-4-chloro-3-hydroxybutyrate to ethyl(R)-4-cyano-3-hydroxy-butyrate with at least 2-fold greater HHDHactivity than the HHDH polypeptide of SEQ ID NO: 2 and which comprisesan amino acid sequence that is at least 98% identical to SEQ ID NO:2040.
 2. The recombinant HHDH polypeptide of claim 1 in which the aminoacid sequence is at least 99% identical to SEQ ID NO:
 2040. 3. Therecombinant HHDH polypeptide of claim 1 or 2 wherein the activity isdetermined under conditions of pH of about 7.0 with 10-100mM ethyl(S)-4-chloro-3-hydroxybutyrate and 500 mM HCN.
 4. The recombinant HHDHpolypeptide of claim 1 , wherein the amino acid sequence is selectedfrom the group consisting of SEQ ID NO:1896, SEQ ID NO:1914, SEQ IDNO:1916, SEQ ID NO:1918, SEQ ID NO:1926, SEQ ID NO:1928, SEQ ID NO:1932,SEQ ID NO:1942, SEQ ID NO:1956, SEQ ID NO:1962, SEQ ID NO:1990, SEQ IDNO:1992, SEQ ID NO:2010, SEQ ID NO:2016, SEQ ID NO:2024, SEQ ID NO:2036,SEQ ID NO:2040, SEQ ID NO:2042, SEQ ID NO:2044, SEQ ID NO:2046, SEQ IDNO:2048, SEQ ID NO:2050, SEQ ID NO:2052, SEQ ID NO:2054, SEQ ID NO:2058,SEQ ID NO:2060, SEQ ID NO:2062, SEQ ID NO:2064, SEQ ID NO:2066, SEQ IDNO:2068, SEQ ID NO:2070, SEQ ID NO:2078, SEQ ID NO:2080, SEQ ID NO:2098,SEQ ID NO:2102, SEQ ID NO:2106, SEQ ID NO:2108, SEQ ID NO:2112, SEQ IDNO:2114, SEQ ID NO:2116, SEQ ID NO:2128, SEQ ID NO:2250, SEQ ID NO:2270,SEQ ID NO:2290, SEQ ID NO:2328, SEQ ID NO:2330, SEQ ID NO:2332, SEQ IDNO:2334, SEQ ID NO:2344, SEQ ID NO:2348, SEQ ID NO:2350, SEQ ID NO:2352,SEQ ID NO:2354, SEQ ID NO:2364, SEQ ID NO:2370, SEQ ID NO:2374, SEQ IDNO:2376, SEQ ID NO:2384, SEQ ID NO:2386, SEQ ID NO:2388, SEQ ID NO:2392,SEQ ID NO:2434, SEQ ID NO:2436, SEQ ID NO:2712, SEQ ID NO:2742, SEQ IDNO:2746, SEQ ID NO:2748, SEQ ID NO:2750, SEQ ID NO:2752, SEQ ID NO:2754,SEQ ID NO:2768, SEQ ID NO:2844, and SEQ ID NO:2848.
 5. The polypeptideof claim 1 , wherein the polypeptide is capable of converting ethyl(S)-4-chloro-3-hydroxybutyrate to ethyl (R)-4-cyano-3-hydroxybutyratewith at least 1.5 fold greater HHDH activity than the HHDH polypeptideof SEQ ID NO: 730, as determined under conditions of pH of about 7.0with 10-100mM ethyl (S)-4-chloro-3-hydroxybutyrate and 500 mM HCN. 6.The polypeptide of claim 1, wherein the polypeptide is further capableof maintaining HHDH activity in the presence of the product ethyl(R)-4-cyano-3-hydroxybutyrate (HN).
 7. The polypeptide of claim 1,wherein the polypeptide is further capable of increased resistance toinhibition by ethyl-4-chloroacetoacetate (ECAA) as compared to thewild-type Agrobacterium sp. HHDH polypeptide of SEQ ID NO:2.
 8. Thepolypeptide of claim 1, wherein the amino acid sequence comprises SEQ IDNO: 2040.